Automated High-Resolution Semi-Preparative Gradient Recycling Liquid Chromatography: Principles, Design, and Applications

A semi-preparative twin-column recycling liquid chromatography (TCRLC) process was extended from isocratic to gradient elution mode. The main separation challenge is when the sample mixture contains early, nearly coeluting, and late impurities, all at the same time. To further improve classical isocratic TCRLC, the gradient TCRLC (GTCRLC) process was implemented with a 2-position 4-port valve in order to better shave the targeted sample from all these impurities. Prior to fully resolving the target compound(s) from the closest impurities by classical isocratic TCRLC, the added valve enabled full elimination by gradient elution mode of not only the early impurities but also any highly retained late impurities that could contaminate the collected fractions of the target compound(s). This GTCRLC process was entirely automated regarding the initial gradient applied, the recycling conditions, and the actuation times of the two valves. The GTCRLC process was applied for the isolation of a single polycyclic aromatic hydrocarbon (PAH), chrysene, present in a complex PAH mixture. In addition, the GTCRLC process was successfully applied to clean vitamins D2 and D3 from a milk extract and to baseline resolve them.