Instrumentation

HPLC

HPLC instrumentation includes a pump, injector, column, detector and recorder or data system, connected as shown below. The heart of the system is the column where separation occurs. Since the stationary phase is composed of micrometer size porous particles, a high pressure pump is required to move the mobile phase through the column. The chromatographic process begins by injecting the solute onto the top of the column. Separation of components occurs as the analytes and mobile phase are pumped through the column. Eventually, each component elutes from the column as a narrow band (or peak) on the recorder. Detection of the eluting components can be either selective or universal, depending upon the detector used. The response of the detector to each component is displayed on a chart recorder or computer screen and is known as a chromatogram.

Retention mechanism
In general, HPLC is a dynamic adsorption process. Analyte molecules, while moving through the porous packing bead, tend to interact with the surface adsorption sites. Depending on the HPLC mode, the different types of the adsorption forces may be included in the retention process:

Hydrophobic (non-specific) interactions are the main ones in reversed-phase separations.
Dipole-dipole (polar) interactions are dominated in normal phase mode.
Ionic interactions are responsible for the retention in ion-exchange chromatography.

All these interactions are competitive. Analyte molecules are competing with the eluent molecules for the adsorption sites. So, the stronger analyte molecules interact with the surface, and the weaker the eluent interaction, the longer analyte will be retained on the surface. SEC (size-exclusion chromatography) is a special case. It is the separation of the mixture by the molecular size of its components.