Mechanism of retention loss when C8 and C18 HPLC columns are used with highly aqueous mobile phases

We describe investigations into the cause of retention losses encountered when C8 and C18 HPLC columns are used with highly aqueous (>90% water) mobile phases. A procedure for quantifying these losses is described, involving stopping and restarting the flow. This procedure was used to study the dependence of retention loss on the pore size, surface concentration, and chemical structure of the bonded phase. Experiments were also carried out to determine how to restore the original retention of the columns by changing the composition of the mobile phase, or by increasing the pressure applied to the column. The results are shown to be consistent with a mechanism based on the theory of pore filling by non-wetting liquids, as employed in Mercury Porosimetry. The retention losses are attributed to the highly aqueous mobile phase being forced out of the pores when the flow is stopped and the pressure released. Retention is lost because the mobile phase is no longer in contact with the interior surface of the particles, where most of the surface area is located. The implications of this phenomenon for maximizing the reversed phase retention of polar analytes are discussed.