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

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. Mass spectrometers

Columns  High-performance liquid chromatography (HPLC) analytical columns are stainless steel tubes, High-performance liquid chromatography (HPLC)  typically of 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.