Liquid chromatography and High

Perfomance Liquid Chromatography

(HPLC)

By
LEBECHI A.K.
 Chromatography
 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
Mikhail Semenovich Tswett

Mixture
Components
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!