Rapid reversed-phase liquid chromatography separation of cyclolinopeptides with monolithic and microparticulate columns

Three monolithic C18-bonded silica gel columns i.e. Chromolith® SpeedROD (CSR), Chromolith® Performance (CP), and Chromolith® High Resolution (CHR), MerckKGaA Darmstadt, Germany and two particle-based columns i.e. ZORBAX Eclipse XDB-C18 (ZEX), Agilent and POROS R1/20 (POR), Applied Biosystems were compared for their performance in separating a mixture of flaxseed cyclolinopeptides (CLs). Gradient mobile phases of acetonitrile and water were optimized for each column. The performance of CHR column in profiling CL standards, measured as the resolution of individual CL, selectivity, and peak asymmetry exceeded the performance of traditional particle-packed columns and the other monolithic columns. The profiling of CLs in aqueous methanolic flaxseed extract was optimized for high-throughput analysis. A total analysis time of 1.5 min at a flow rate of 3.0 mL min−1 was achieved on a CSR column. Injection of over 2000 methanol extracts of flaxseed on a CSR column had no impact on backpressure or resolution of a standard CL mixture.

► Comparison of reverse phase monolithic and perfusion HPLC columns.
► An optimized gradient was developed to separate flaxseed cyclolinopeptides.
► Gradient elution with total analysis time of 1.5 min was achieved.
► Monolithic columns offer constant resolution and back pressure over 2000 samples.
► Monolithic column is suitable for high-throughput screening of cyclolinopeptides.