9. Theory of Separation

The principle of liquid chromatography separation is based on the distribution of the sample between the mobile and stationary phase (column material). The higher the affinity of the sample to the stationary phase (more interaction), the longer it takes for the individual components to go through the column. The compounds that interact the most, leave the column the latest, hence have the longest retention time.

Retention time = the time between the injection of the sample and its detection in the detector. The more the compound interacts with the column packing material, the longer it is retained in the column, and hence has the longer retention time/takes longer to reach the detector. Under standardized conditions, this retention time is reproducible (always the same) and hence used for the identification of the compound.

Attached is a document that goes into detail on the chromatographic separation theory.