The HPLC operating conditions using a methanol-water gradient have been widely applied by many researchers, allowing the separation of good quality peaks on the chromatogram [ 1, 24].
Although a methanol gradient was used in the HPLC experiment (12.5% methanol at 10 min retention time), the relative peak intensities are comparable with those obtained with the direct injections.
Gonads with mesonephroi attached were rehydrated through a methanol gradient then wash with PTW.
Retinoids were separated on a reverse phase pro-C18 column (250 × 4.6 mm; 12 nm, S-5 μm; YMC Europe, Dinslaken, Germany) using a gradient of methanol:toluene at a flow rate of 1.0 ml/min.
The substrate ATP and the product ADP were separated on a C-18-silica column using a binary gradient of Methanol and the ion pairing agent TBAHS (10 mM, pH = 6) as the mobile phase, 50 ul volume of standard PPK reaction was injected and separation monitored at 260 nm at a flow-rate of 1 ml/min.
Fraction E (18.08 g possessed the highest antioxidant and anti-hepatocarcinoma activity and was consecutively re-chromatographed on SephadexLH-20 (5 × 70 cm, 760 g) using a gradient of methanol-H2O (2700 mL) 100 0,80 20,60 40,40 60,20 80.
dUMP formed in the course of the reaction was eluted using a linear gradient of 5% methanol in 15 mM Triethylamine pH 7.9 (0.6 mL.min-1) and detected at 260 nm using a diode array detector (Waters 2696 PDA detector).
Liquid chromatography was performed with a Waters HSS C18 column (1.0 mm i.d. ×50 mm, 1.8-um particles) using a linear gradient of 0% to 100% methanol in 0.1% aqueous ammonium formate, pH 6.0.
For PLM D1, the reaction mixture was separated using a solvent gradient of solvent C and methanol: 20% methanol in solvent C for 5 min, 20 to 80% methanol for 17 min, and 80 to 100% methanol for 1 min. Acetylated Cu II -PLM D1 was Cu II -PLM pooleD1 and subjected to HR-MALDI-MS analysis, yielding the [M]+ ion at m/ z 1531.511 (calculated [M]+ ion for C57H88N20O22S2Cu at m/ z 1531.512).
The valves were switched, and the analytes were backwashed onto a reversed-phase or strong anion exchange analytic column and separated using a linear gradient beginning with 10% methanol in 0.1% formic acid and ending with 100% methanol.
