High Performance Liquid Chromatography

 

High Performance Liquid Chromatography

High Performance Liquid Chromatography

Ø  A form of column chromatography to separate, identify and quantify different components of a mixture

Ø  Developed in 1970

Ø  The most widely used analytical technique

Ø  HPLC is an extension of conventional liquid chromatography.

Ø  Powerful tool in analytical techniques

Ø  Columns are tightly packed, and the eluent is forced through the column under high pressure ( up to 5000 psi) by a pump.

Ø  Allows to use a very smaller particle size of the column packing material which gives a much greater surface for interactions between the stationary phase and the molecules flowing through it

Ø  Allows a much better separation of the component of the mixture.

HPLC technique

Ø  Utilize liquid mobile phase to separate the mixture

Ø  Analytes are 1^st dissolved in a solvent then through column applying high pressure.

Ø  Mixture is resolved into their respective component on the basis of retention time in the column.

Components of HPLC

Ø  Pump

Ø  Injector

Ø  Column

Ø  Detector

Ø  Data record system

Pump

Ø  A pump forces the mobile phase trough the column at a much greater speed than gravity-flow columns

Ø  Pumps are designed in order to maintain a stable flow rate.

Ø  Flow range 0.01 to 10 ml/min

Injector

Ø  Inject the liquid sample within range of 0.1 to 200 micron

Column

Ø  Smooth-bore stainless steel or heavy walled glass tubing

Ø  Hundreds of packed columns differing in size and packing are available.

Ø  Columns packing very in size from 0.3 to 20 micron.

Ø  The most commonly used packing material is silica gel.

Detector

Ø  Hplc detector monitor the elute as it leaves the column.

Ø  Produce electronic signals proportional to the concentration of each separated component.

Types of Detector

Ø  UV detector

Ø  Refractive index detector

Ø  PDA detector

Ø  Electrochemical detector

Ø  Fluorescent detector

Ø  Mass spectrophotometer

Classification on the basis of polarity of mobile phase and reverse phase

Ø  Normal phase

     non polar mobile phase and polar stationary phase

Ø  Reverse phase

     polar mobile phase and non polar stationary phase

Applications

Ø  Chemistry and biochemistry research analyzing complex mixtures.

Ø  Purifying chemical compound.

Ø  Quality control to ensure the purity of raw material.

Ø  Analyzing air and water pollutants.

Ø  Monitoring pesticide level in the environment.

Ø  To survey food and drug products.

Ø  To identify confiscated narcotics.

Ø  To determine the amount of impurities in newly drug products.

Ø  Isolation of natural products.

Ø  Developing processes for synthesizing chemical compound.