Introduction

High-performance liquid chromatography (HPLC) is a form of liquid chromatography .

The main purpose is to separate compounds that are dissolved in solution.

HPLC instruments consist of:a.a reservoir of mobile phase,b. a pump, c.an injector, d.a separation column, e.and a detector.

Compounds are separated by injecting a plug of the sample mixture onto the column.

The different components in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase

Instrumentation

Solvents must be degassed to eliminate formation of bubbles.

The pumps provide a steady high pressure with no pulsating, and can be programmed to vary the composition of the solvent during the course of the separation

The liquid sample is introduced into a sample loop of an injector with a syringe.

The presence of analytes in the column effluent is recorded by :

a.detecting a change in refractive index, b.UV absorption at a set wavelength, c.fluorescence after excitation with a

suitable wavelength, d.or electrochemical response

Mass spectrometers can also be interfaced with liquid chromatography to provide structural information and help identify the separated analytes.

Columns

High-performance liquid chromatography (HPLC) analytical columns are stainless steel tubes,

typically of 10-30 cm in length and 3-5 mm inner diameter.

Guard columns, which are placed before an analytical column to trap junk and extend the lifetime of the analytical column, are 3-10 cm long

Types

Partition Chromatography In partition chromatography the

stationary phase is bonded to inert particles of 3-10 mm of diameter,

Analytes separate as they travel through the column due to the differences in their partitioning between the mobile phase and the stationary phase

Reverse-phase partition chromatography:

uses a relatively nonpolar stationary phase and a polar mobile phase, such as methanol, acetonitrile, water, or mixtures of these solvents.

Reverse-phase chromatography is the most common form of liquid chromatography, primarily due to the wide range on analytes that can dissolve in the mobile phase

Normal-phase partition chromatography:

uses a polar stationary phase and a nonpolar organic solvent, such as n-hexane, methylene chloride, or chloroform, as the mobile phase. The stationary phase is a bonded siloxane with a polar functional group.

Adsorption Chromatography: The stationary phase are silica or

alumina particles. Analytes are separated due to their varying degree of adsorption onto the solid surfaces.

Ion Chromatography Cation and anion-exchange resins

are used.