The procedure of FPLC depends on the type of chromatography that users want to perform. However, the basic procedure is the same.
1. In FPLC is essential to keep constant the flow rate of the solvents (buffers). This is set and control by pumps which conduce the solvents to the mixer.
2. The mixer combines the buffers used in the process and elutes a unique solution with different concentrations of the buffers. This depends of the stage of the process, increasing the concentration of the buffer B from 0% at the beginning of the procedure to 100% at the end.
3. Next, the sample is injected through the injection valve and then carried into the column by the flowing solvent from the mixer.
4. Once in the column, the sample mixture separates as a result of different components adhering to or diffusing into the stationary phase.
5. The buffers are lead to the bottom of the column by the flow rate. On the other hand, the sample is separated in different components. This is resulted by the separation of components by the size, charge, affinity and solubility of the components of the sample.
6. After this, the elution is carried to the UV monitor where the conductivity (salt concentration), pH and UV absorbance (proteins concentration) is measured.
7. Finally, the elution is collected by the fraction collector and the monitor or recorder can show the data obtained.