The AF4 experiment can be separated into three stages:
1. Sample Injection
:Samples are injected into the system using a known amount of sample volume. This volume will depend on the AF4 instrument being utilized in the experiment. Starting fractionation immediately after sample injection is not ideal because the sample is going to spread out randomly from the injection site, so the beginning velocity and place of the particles are not all the same. This leads to line broadening and insufficiency. In order to correct such an error, sample focusing is proposed.
2. Sample focusing<br />
:A current going opposite of the carrier solvent is used to focus all the particles in the sample to one specified area before fractionation begins. This corrects for any peak broadening that can occur due to particles being dispersed from the injection port to the channel outlet before fractionation begins. Sample preparation is another option that can be achieved in the focus step. Once all the particles are in the same area of the channel, fractionation can occur.
3. Fractionation<br />
:There are two components that make up the FFF system. Firstly, the laminar flow that carries the sample through the separation chamber and secondly the separation field applied perpendicular to the channel, against the sample flow.
:As particles flow along the channel the cross flow separation field pushes the molecules towards the bottom of the channel. As they pass by the bottom they undergo a counter acting diffusion back into the channel against the carrier flow. The extent to which the molecules can diffuse back into the channel is dictated by their natural Brownian motion, a characteristic based on size that is unique to each individual species. Smaller particles have a higher Brownian motion than larger ones and are able to diffuse higher into the channel against the carrier flow.
:The rate of laminar flow within the channel is not uniform. It travels in a parabolic pattern with the speed of the flow increasing towards the center of the channel and decreasing towards the sides. Therefore, the rate at which particles will be carried through will depend on their position within the channel. Those with a greater diffusion, located in the center of the channel, will be transported with a greater velocity. The larger particles in the shallow, slower moving stream are transported with lower flow velocity and elute later than smaller particles. This results in a gentle separation of particles based on mass with the elution order of smallest to largest.