Introduction: Typical gradient LC methods include two segments: the gradient in which the separation occurs, and the regeneration step where the column is washed with strong solvent and re-equilibrated to initial conditions, ready for subsequent sample injections. Washing with 3-5 column volumes of a suitable strong solvent is usually recommended, as this prevents accumulation of phospholipids and potentially extends the useful life of the LC column. Allowing sufficient re-equilibration time is important for method robustness. Reducing the time allowed for regeneration may produce acceptably performing high-throughput methods in some cases, but it comes with the risk of poor-quality data and limiting column longevity.

There is an alternative hardware configuration, parallel column regeneration, which splits the elution gradient and regeneration phases over two consecutive injections, allowing sufficient washing and re-equilibration to be performed on the ‘passive’ column, while the gradient separation occurs on the ‘active’ column. This has the potential to dramatically shorten the analytical run, increase sample throughput, without compromising UPLC best practices.