High Performance Liquid Chromato: School of Basic and Sciences
High Performance Liquid Chromato: School of Basic and Sciences
High Performance Liquid Chromato: School of Basic and Sciences
Department of chemistry
High Performance Liquid
Chromatography
Presentation by
Maruthanayagam. R
Register No. D190504
PG Diploma in Chemical Lab Technician
Central University of Tamil Nadu
Evolution of HPLC
• 1903 - The Russian botanist Mikhail Tsvet is considered to have
‘invented’ the chromatographic technique when he reported
separations of different plant pigments into a series of colored bands
on a packed column. He called this technique ‘chromatography’.
• The origins of HPLC date back to the invention of chromatography in
the early 20th century, through the introduction of partition and
paper chromatography in the 1940s, to the introduction of liquid
chromatography in the early 1960s. Shortly thereafter, the need for
better resolution and high-speed analyses of nonvolatile samples led
to the development of HPLC.
• 1969 - The first commercial HPLC was manufactured by
Waters Corporation, and was known as the ALC100 HPLC.
Jack Kirkland (left), with technician Glen Wallace, in front of a homebuilt HPLC instrument in 1967.
Introduction
• HPLC stands for“High-performance liquid chromatography”(sometimes referred to
as High- pressure liquid chromatography).
Solvent
reservoirs
1 and degassing
2
5
3
4
1 – Pump
2 – Injector
3 – Column
4 – Detector
5 – Computer
Picture of HPLC instrument
Pump:
• The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatograph at a specific flow rate, expressed in
milliliters per min (mL /min).
• Normal flow rates in HPLC are in the 1-to 2-mL/min range.
• Typical pumps can reach pressures in the range of 6000-9000 psi (400-
to 600-bar).
• During the chromatographic experiment, a pump can deliver a
constant mobile phase composition (isocratic) or an increasing
mobile phase composition (gradient).
Pump Module–types:
• Isocratic pump - Delivers constant mobile phase composition;
• solvent must be pre-mixed;
• lowest cost pump
• Gradient pump - Delivers variable mobile phase composition;
• can be used to mix and deliver an isocratic mobile phase or a gradient mobile phase
–Binary gradient pump –delivers two solvents
• The injector serves to introduce the liquid sample into the flow stream
of the mobile phase.
• The injector must also be able to withstand the high pressures of the liquid
system.
• An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical .
Manual Injector:
1. User manually loads sample into the injector using a syringe
2. and then turns the handle to inject sample into the flowing mobile
phase which transports the sample into the beginning (head) of the
column, which is at high pressure
Auto sampler:
3. User loads vials filled with sample solution into the auto sampler tray
(100 samples)
4. and the auto sampler automatically
a) measures the appropriate sample volume,
b) injects the sample,
c) then flushes the injector to be ready for the next sample,
etc., until all sample vials are processed for unattended
automatic operation
Manual Injectors
Sample Loop
Load - Inject
Inject
Automatic Injectors
Step 1 Step 2
Step 3
HPLC Column:
Packing material:
• The packing material is prepared from SILICA particle, ALUMINA particle and ion exchange
RESIN.
• Porous plug of stainless steel or Teflon are used in the end of the columns to retain the
packing material.
• According to the mode of HPLC , they are available in different size , diameters, pore size or
they can have special materials attached ( such as antigen or antibody ) for immuno affinity
chromatography.
Column types
• Normal phase
• Reverse phase
• Size exclusion
• Ion exchange
Normal phase STATIONARY PHASES
(NORMAL POLARITY)
• In this column type, the Silica or alumina possess polar sites that
retention is governed by interact with polar molecules.
the interaction of the silica
polar parts of the O
stationary phase and Polar Group HO Si
solute. O
Mass Spectrometry
•The mass spectrometry detector coupled with HPLC is called HPLCMS. HPLC-MS is
the most powerful detector,widely used in pharmaceutical laboratories and research
and development.
•The principal benefit of HPLC-MS is that it is capable of analyzing and providing
molecular identity of a wide range of components.
Data processing unit (Computer)
• Frequently called the data system, the computer not only controls all
the modules of the HPLC instrument but it takes the signal from the
detector and uses it to determine the time of elution (retention time)
of the sample components (qualitative analysis) and the amount of
sample (quantitative analysis).
• The concentration of each detected component is calculated from the
area or height of the corresponding peak and reported.
Applications
HPLC is one of the most widely applied analytical separation
techniques.
Pharmaceutical:
• Tablet dissolution of pharmaceutical dosages.
• Shelf life determinations of pharmaceutical products.
• Identification of counterfeit drug products.
• pharmaceutical quality control.
Environmental:
• Phenols in Drinking Water.
• Biomonitering of PAH pollution in high-altitude mountain lakes through the analysis of fish
bile.