AFFF is a chromatography-like technique introduced by Giddings in 1965 that separates nanoscale analytes based on their diffusion behavior instead of chemical interaction with the separation column. A Wyatt Eclipse AFFF was connected with a pump, an autosampler, and the separation channel. All fractionation methods consist of five general steps: equilibration, injection, focus, separation, and washing. During the pre-separation, the system is flushed with 200 ppm sodium azide in deionized water as an eluent. Then, 50 µL of the analyte is injected into the channel and focused in the separation area of the channel. Different diffusion coefficients lead to different channel positions and elution speeds: the larger the particles, the longer the retention time. After the separation, a washing step is performed to remove sample residues. A 10 kDa PES membrane is used as an accumulation membrane with a 350 µm spacer. The separation method was optimized following a workflow proposed by Gigault et al. in 2014. The crossflow Vx used in the AFFF separation method (Table ) varies depending on the separation method applied. Either a constant, a linearly decreasing, or an exponentially decreasing Vx is applied.