Biotage reversed phase flash purification whitepaper

A flash column’s loading capacity varies depending on several factors but primarily the selectivity and resolution of the target compound from its closest eluting neighbors. While normal-phase flash chromatography loading capacity is commonly determined from thin-layer chromatography (TLC) separation data, reversed-phase loading capacity determination is typically an empirical process involving several repeated injections with increasing sample mass.

As a general rule, reversed-phase columns have lower loading capacity compared to normal-phase silica columns due to reversed-phase media’s lower available surface area (bonding C18 to silica reduces surface area) and different separation mechanisms (partitioning vs. silica adsorption/desorption). If you check various flash column vendors you will find silica loading capacities up to 10% and even 20% of media weight. In contrast, typical published reversed-phase capacities are 1% - 2% of media weight. These are suggested maximum loads and not necessarily meant to be average or applicable for every purification. Again, it depends on how good your separation is along with the other criteria mentioned above.

In this document we will show how using a resolution value (Rs), calculated from small-scale empirical scouting runs, can be used to suggest the maximum sample load mass possible while maintaining a specific product purity goal.