Liquid chromatography and High Perfomance Liquid Chromatography

(HPLC)

By LEBECHI A.K.

Chromatography

Mikhail Semenovich Tswett

Chromatography was discovered in 1903 and was used to separated plant pigments.

It is a popular method used to separate mixtures to their individual components.

Separate

Components Mixture

TYPES OF CHROMATOGRAPHIC TECHNIQUES

Thin layer chromatography

COLUMN CHROMATOGRAPHY

HPLC The history of HPLC began in the 60’s and was known as high

pressure liquid chromatography.

The instrumentation and columns improved over the time and it is now known as high performance liquid chromatography.

HPLC separation technique provides high speed, efficiency and high

sensitivity as compared to liquid chromatography.

A very small volume of sample is injected into a tube packed with a stationary phase (3-5 microns particles).

The sample is pushed down the column with a mobile phase by applying high pressure and the components are detected and quantified as they exit the column.

Components of HPLC 1. Pump: pumps the mobile phase at a specific flow rate in mL/min. The

pump pressure is normally between 400-600 bar. 1. Injector: Introduces the sample into the column (about 5-20 μL). 1. Column: provides separation through high pressure created by the small

particles.

2. Detector: it quantifies and identify the sample components and provides information to the computer.

3. Computer: takes the signals from the detector and displays the retention times and quantity of the components.

HPLC system

Separation columns Normal phase: Column packing is polar (e.g silica) and the mobile phase is non

polar. It is used for water-sensitive compounds, geometric isomers, cis-trans isomers and chiral compounds.

Reverse phase: the column packing is non-polar (e.g C18) , mobile phase is water+ miscible solvent (e.g methanol). It can be used for polar, non polar, ionizable and ionic samples.

Ion exchange: Column packing contains ionic groups and the mobile phase is buffer. It is used to separate anions and cations.

Size exclusion: molecules diffuse into pores of a porous medium and are separated according to their relative size to the pore size. Large molecules elute first and smaller molecules elute later.

General applications HPLC is used in diagnosis of diseases and disorders. It is used to estimate metabolites in urine samples and plasma. For quality assurance purposes in both food and pharmaceutical

industries. In scientific research for analysis.

Conclusions

Chromatography is an effective method of purification.

The use of HPLC as a support method of analysis could explain the fluorescence behavior of the compounds.

HPLC is not for decoration, lets be more analytical!