High-Performance Liquid Chromatography: Introduction

In high-performance liquid chromatography or HPLC, separation occurs in an analytical column packed with a silica-based, bonded stationary phase.

The separation of analytes takes place based on the interactions between the solid stationary phase and the liquid mobile phase.

The silica surface also consists of unreacted silanol groups, which causes polar interactions that leads to tailing.

So, silanol groups are reacted with trimethylchlorosilane to form the end-capped stationary phase.

A guard column packed with the same stationary phase is placed before the analytical column to protect it from contamination and is replaced regularly to increase the column's lifetime.

The polarity of the mobile phase plays a crucial role in the elution of analytes.

Normal-phase chromatography uses a polar stationary phase and a nonpolar mobile phase.

In contrast, reverse-phase chromatography uses a nonpolar stationary phase and a polar mobile phase.