AIM

To demonstrate the basic principles of high performance liquid chromatography in qualitative

and quantitative analysis.

INTRODUCTION
High-performance liquid chromatography (HPLC) is a separation technique that can be used for
the analysis of organic molecules and ions. It uses the mechanisms of adsorption, partition and
ion exchange, all depending on the type of stationary phase used. HPLC involves a solid
stationary phase, normally packed inside a stainless-steel column, and a liquid mobile phase.
Separation of the components of a solution results from the difference in the relative distribution
ratios of the solutes between the two phases.
In this experiment, reversed phase HPLC is applied. In reversed phase chromatography,
hydrophobic molecules are adsorbed onto a hydrophobic solid support in a polar mobile phase. If
more organic solvent is added, it reduces the hydrophobic interaction between the solute and the
solid support resulting in de-sorption due to the decrease in mobile phase polarity. The more
hydrophobic the molecule the more time it will spend on the solid support and the higher the
concentration of organic solvent that is required to promote de-sorption.
In recent times, because it is more reproducible and has broad applicability, reversed-phase
chromatography is used for approximately 75% of all HPLC methods. Most of these protocols
use as the mobile phase an aqueous blend of water with a miscible, polar organic solvent, such as
acetonitrile or methanol. This typically ensures the proper interaction of analytes with the nonpolar, hydrophobic particle surface. A C18bonded is the most popular type of reversed-phase
HPLC packing.
Reversed phase chromatography is the most popular separation technique at analytical scale
because it applies to a very wide range of molecules including charged and polar molecules and
is a robust technique. Besides that, it allows precise control of variables such as organic solvent
type and concentration, pH, and temperature. The columns used in reversed phase
chromatography are also efficient and stable.