Increasing the resolution of field flow fractionation with increasing crossflow gradients

The resolution of flow field flow fractionation (flow FFF) depends primarily on the crossflow rate and its change over time. In this work we demonstrate a method for modulation of the crossflow rate during separation increases the peak-to-peak resolution of the resulting fractograms. In classical FFF methods, the crossflow rate is either maintained constant or decreased during separation of the different species. In this work, higher resolution between peaks was achieved by a novel gradient method in which the crossflow is increased briefly during separation to allow stronger retention of the later eluting peaks. We first outline the theoretical basis by which the improved separation is achieved. We confirm our hypothesis by quantifying the impact of increasing crossflow on the resolution between a monoclonal antibody monomer and its high molecular weight (HMW) aggregate. We then demonstrate that this method is applicable to two different FFF methods (AF4 and HF5) and various pharmaceutically relevant samples (monoclonal antibodies (mAbs) and adeno-associated viruses (AAV)). Finally, we hypothesize that increasing the force perpendicular to laminar flow as described here is broadly applicable to all FFF methods and improve the quality of FFF-based separations.