High-performance liquid chromatography (HPLC) is a popular analytical technique used to separate, identify, and quantify mixtures into their individual components. HPLC uses high pressure pumps to push a pressurized mobile phase through a column packed with adsorbent particles. Sample components interact differently with the stationary phase based on their properties and are eluted from the column at different retention times, allowing for separation. The separated components are then detected and quantified to analyze the sample. HPLC provides high-speed, efficient, and sensitive separation compared to traditional liquid chromatography.

1. High-Performance
Liquid Chromatography
Prepared by- ALOK RANJAN
(M.V.Sc. II YEAR)
Deptt. Of Animal Nutrition

2. 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.

4. Scope
• 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.

5. Introduction
• A popular analytical technique used for the separation,
identification and quantification
• solvent usually flows through column but here,solvents
are forced.
• solvent will be forced under high pressures upto 400
atmospheres so that sample can be separated
• Chromatography can be depicted as a mass exchange
process including adsorption
• the adsorbent, is regularly a granular material made of
solid particles (e.g. silica, polymers, etc.) 2 μm to 50 μm
in size
• pressurized fluid is commonly a blend of solvents (e.g.
water, acetonitrile and/or methanol) and is known as
‘mobile phase’

6. • (HPLC depends on pumps to pass a pressurized fluid and
an example blend through a section loaded with
adsorbent, prompting the partition of the specimen
segments.)
• -The pressurized fluid is commonly a blend of solvents
(e.g. water, acetonitrile and/or methanol) and is known
as ‘mobile phase’.
• Its organization and temperature plays an important part
in the partition procedure by affecting the connections
occurring between sample segments and adsorbent

7. 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.
2. Injector: Introduces the sample into the column (about 5-20
μL).
3. Column: provides separation through high pressure created
by the small particles.
4. Detector: it quantifies and identify the sample components
and provides information to the computer.
5. Computer: takes the signals from the detector and displays
the retention times and quantity of the components.

8. Principle of HPLC
• The process involves interaction of compounds in the analyte
(which travels along the mobile phase ) across an immobile
surface(stationary phase).
• The compounds bind at the specific regions of stationary
phase based on certain physical and chemical properties.
• This bound molecules are then eluted with a suitable buffer
and are the same collected with time.
• The more polar compounds adsorbed with silica i.e. stationary
phase, Hence move slowly while compounds with
hydrophobic nature doesn’t bind with stationary phase, Hence
moves faster & comes out first and displays its graph.