High performance liquid chromatography (HPLC) is a technique used to separate components in a mixture. It works by differential migration of solutes through a column containing a stationary phase as a pressurized mobile phase flows through. Solutes are detected as they elute from the column. Key aspects of HPLC include the use of a high pressure pump to deliver the mobile phase, columns packed with microparticulate stationary phases, and detectors to identify eluted components. HPLC is useful for separating compounds that are not volatile enough for gas chromatography.

1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)
Chromatography???
IUPAC has drafted a recommended
definition of chromatography: ‘
Chromatography is a physical method
of separation in which the components
to be separated are distributed
between two phases, one the which is
stationary phase, while other is mobile
phase moves in a definite direction’’
High-performance liquid chromatography (HPLC) is a technique
for the separation of components of mixtures by differential
migration through a column containing a microparticulate solid
stationary phase. Solutes are transported through the column by
a pressurized flow of liquid mobile phase, and are detected as
they are eluted.
Basic Vocabulary
 Mobile Phase
 Solvent Delivery
System
 Column and
Stationary Phase
 Comparison with
GC
Why need HPLC????

2. Need due to
Because approximately 85% of known compounds are
not sufficiently volatile or stable to be separated by GC.
Transformation from High Pressure Liquid
Chromatography to High Performance Liquid
Chromatography.
In 1964 Calvin Giddings from University of Utach Published
a Paper Entitled ‘Comparison of the Theoretical limit of
Separation Ability in Gas and Liquid Chromatography’’
He Said in his prediction
‘’Improve LC Performance if one could use small particles
under Increased flow Pressure’’
Csaba Horvath and S.R Lipsky built first HPLC and called
‘High Pressure Liquid Chromatography’’ after 1970s
technology produces Small Silanized Silica particles
allowed to used small volume longer column necessary to
give the high resolution performance. Today HPLC become
known as HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY.

3. Mobile Phase
The mobile phase is either a single solvent or a blend of two
or more having the appropriate eluting power for the
sample components. It ranges from a nonpolar liquid to
aqueous buffers mixed with an organic solvent.
Solvent Delivery System
The solvent delivery system comprises a means of
degassing, filtering and blending up to four solvents which
are then delivered to the top of the column under pressure
by a constant flow pump.
Sample Injection
Liquid samples or solutions are introduced into the flowing
mobile phase at the top of the column through a constant or
variable volume loop and valve injector that is loaded with a
syringe
Column and Stationary Phase
Columns are straight lengths of stainless steel tubing tightly
packed with a microparticulate stationary phase. The
column packings are chemically modified
silicas, unmodified silica or polymeric resins or gels.

4. Mobile Phase Reservoirs and Solvent Treatment System
Mobile phase serves to transport the sample to the system.
Essential criteria of mobile phase are inertness to the sample
components. Pure solvents or buffer combinations are
commonly used. The mobile phase should be free of
particulate impurities and degassed before use.
Sparging is a process in which dissolved gases are swept
out of a solvent by bubbles of an inert, insoluble gas.
Isocratic Elution in HPLC is one in which the solvent
composition remain same.
Gradient Elution in HPLC is one in which the composition
of the solvent is changed continuously or in a series of steps.

5. Pumping System
General Requirement;-
 Ability to Generate pressure of up to 6000 psi
 Pulse Free Output
 Flow rates ranging from 0.1 to 10 mL/min
 Flow reproducibility of 0.5% relative or better
 Resistance to corrosion by variety of Solvent
Three Major Types of Pump
 Screw Driven Pump
 Pneumatic or Constant Pressure pump
 Reciprocating Pump
The pump is a
reciprocating piston pump
In this pump, a small piston (approximately ¼ in. in diameter)
is driven back and forth drawing liquid in through an inlet
check valve during its backward stroke and expelling the liquid
through an outlet check valve during the forward stroke. A
check valve is a device that allows liquid flow in one direction
only.

6. Sample Injection
Liquid samples and solutions are injected directly into
the pressurized flowing mobile phase just ahead of the
column using a stainless steel and Teflon valve
fitted with an internal or external sample loop.
Guard Column
A short length column is positioned before the
analytical column which increases the life of
Analytical column by removing particulate matters
and contamination from the solvent.
Column and Stationary Phase
Straight lengths of stainless steel tube, come in various
diameter depending upon the particular application.
Usually I.D 3.9 to 4.6 mm. Length 10 to 30 cm.
Stationary Phases
Mircoporous or Diffusive Particles
Silination Reaction (Chemically Bonded)

7. Most Importantly!!!
Mode of Chromatography
In Normal phase Chromatography (NPC), the
stationary phase is polar. A nonpolar mobile is used.
In Reversed phase Chromatography, a relatively
nonpolar stationary phase is used, with a polar mobile
phase.
Detectors
UV Absorption

8. Diode Array
Fluorescence Detector

9. Comparison with GC
Because the HPLC mobile phase is a liquid, there are
some very obvious differences between HPLC and
GC.
First, the mechanism of separation in HPLC involves the
specific interaction of the mixture components with a
specific mobile phase composition, while in GC the vapor
pressure of the components and not their interaction with
a specific carrier gas, is the most important consideration
Second, the force that sustains the flow of the mobile
phase is that of a high-pressure pump, rather than
the regulated pressure from a compressed gas cylinder.
Third, the injection device requires a totally
different design due to the high pressure of the system
and the possibility that a liquid mobile phase may
chemically attack a rubber septum. Fourth, the detector
requires a totally different design because the
mobile phase is a liquid.
Finally, the injector, column, and detector need not be
heated as in GC, although the mode of separation
occurring in the column can be affected by temperature
changes, and thus sometimes elevated column
temperatures are used.
Muhammad Shakaib Qureshi
Msc Analytical Chemistry
Institute of Chemistry (SALU-KHP)