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.
