How AF4 works?

How AF4 works?

AF4 principle The particle-containing carrier fluid flows parallel to the membrane, but a constriction at the end of the channel forces some of the fluid to pass through the membrane, creating a “cross-flow field” that concentrates the particles towards the membrane.

What field is applied in field flow fractionation?

Different separation forces Depending on the Field-Flow Fractionation method which is used, different force fields (liquid flows, centrifugal forces, temperature gradients or gravity fields) are used and applied perpendicular to the separation channel.

How is AF4 technology used to separate nanoparticles?

We describe the protocol development and optimization of asymmetric-flow field-flow fractionation (AF4) technology for separating and characterizing extracellular nanoparticles (ENPs), particularly small extracellular vesicles (sEVs), known as exosomes, and even smaller novel nanoparticles, known as …

How long does it take for AF4 fractionation?

Although the whole procedure of cell culture and ENP isolation from the conditioned medium by ultracentrifugation (UC) can take ~3 d, the AF4 fractionation step takes only 1 h. Users of this technology will require expertise in the working principle of AF4 to operate and customize protocol applications.

How are fluorescence detectors used in AF4 analysis?

Similar to HP-SEC analysis, in AF4 analysis UV, refractive index and fluorescence detectors are usually used as concentration determining/quantifying detection systems. Additionally, multi-angle laser light scattering (MALLS) detection is used to determine the molecular weight and size of separated species.

When to use AF4 in forced degradation studies?

AF4 is often used in the context of forced degradation studies and real-time stability studies, since those often imply the formation of relatively large amounts of aggregates in the size range covered by AF4.