The best separation was obtained with a mobile phase containing 50 mM phosphate, pH 5.5 and 6% methanol.
Chromatography was performed with mobile phase containing a mixture of acetonitrile and water (50 50, v/v) with flow rate was of 1.0 ml min−1.
Samples were eluted with a mobile phase containing acetonitrile methanol tetrahydrofuran 20 mM K2HPO4 (pH 7.0) (20 10 946946, v/v), and the flow rate of the mobile phase was 1 ml/min.
However, for the neuromedin-like peptides of interest, the C18+ column in combination with a mobile phase containing methanol as organic modifier and acetic acid as additive provided narrower and higher peaks.
Similarly, at a flow rate of 1 mL/min and 10 s collection times, the system precisely dispensed mobile phase containing a [14C]-radiolabeled compound across an entire 96-well plate (% CV was within 5.3%).
Aliquots of 40 μL supernatant were injected onto a C18 reverse-phase high performance liquid chromatography (HPLC) column using a mobile phase containing 60 mM potassium dihydrogen phosphate (KH2PO4, Sigma-Aldrich) and 5 mM tetrabutylammonium chloride (Sigma-Aldrich), pH 6.0, in 30% methanol.
Mobile phase containing 75% methanol and LiChrospher® 100 RP-18, 150 mm column were finally chosen.
Following termination of fermentation and extract preparation using above mentioned protocol, TLC profiles of the both the extracts were compared in mobile phase containing 5% MeOH in DCM.
A C8 chromatographic column (PerkinElmer, America) was used with the mobile phase containing 1 mM tetrabutylammonium hydroxide, 0.05 mM dipotassium EDTA, and 0.05% methanol (pH 6.8).
Chromatogrpahic separation by RPLC was achieved using a reversed-phase column and a water/acetonitrile mobile phase, containing 0.05% formic acid and using a gradient system.
A satisfactory separation of FEX and its four related impurities with satisfactory resolution and increased speed was obtained with a mobile phase containing 40% methanol.
