High Performance Liquid Chromatography

1. High Performance Liquid
Chromatography
Name: Payal Vaswani
Molecular Oncology Laboratory

2. Chromatography
• Mikhail Tsvet invented
the chromatographic
technique.
• Chromatography is a
physical method of
separation that
distributes components
to separate between
two phases, one
stationary phase, the
other moving in a
definite direction(mobile

3. Types of
Chromatography
Chromatographic
bed
Gas chromatography
Paper and Thin layer
chromatography
Physical state of
mobile phase
Gas chromatography
Liquid
chromatography
Separation
mechanism
Size exclusion
Ion exchange
chromatography

4.  Liquid chromatographic systems were largely inefficient due to
the flow rate of solvents being dependent on gravity which lead
to many hours for separation.
 Gas phase separation and analysis of very polar high molecular
weight biopolymers was impossible.
 Following on the seminal work of Martin and Synge in 1941, it
was predicted by Cal Giddings, Josef Huber, and others in the
1960s that LC could be operated in the high-efficiency mode.
 Prof. Csaba Hovarth in 1970 indicated the fact that high
pressure was used to generate the flow required for liquid
chromatography in packed columns.

5.  High Performance Liquid Chromatography (HPLC) is a
form of liquid chromatography to separate compounds that
are dissolved in solution.
 Separation of a sample into its constituent parts because of
the difference in the relative affinities of different
molecules for the mobile phase and the stationary phase
used in the separation.

6. Instrumentation
■Solvent Reservoirs
■Pump System
■Sample Injector
■Temperature Controller
■Columns
■Detectors
■Data System

7.  Mobile phase contents are contained in a
glass reservoir.
 The mobile phase, or solvent, in HPLC
is usually a mixture of polar and non-
polar liquid components whose
respective concentrations are varied
depending on the composition of the
sample.

8.  The role of the pump is to propel a liquid through
the chromatography system at a specific flow rate,
expressed in ml/min.
 Normal flow rates in HPLC are in the 1-2 mL/min
range.
 Typical pumps can reach pressures in the range of
6000-9000 psi.
 Binary gradient pump – delivers two solvents
 Quaternary gradient pump – delivers four solvents

10. ISOCRATIC MODE
 A separation in which the
mobile phase composition
remains constant
throughout the procedure is
termed isocratic.
 Best for simple separations
 Often used in quality
control applications that
support to a manufacturing
process.
GRADIENT MODE
■ A separation in which the
mobile phase composition is
changed during the
separation process is
described as a gradient
elution.
 Best for the analysis of
complex samples
 Often used in method
development for unknown
mixtures.

11.  Injectors are used to provide
constant volume injection of
sample into the mobile phase
stream. Inertness and
reproducibility of injection are
necessary to maintain high level
of accuracy.
 An injector for an HPLC system
should provide injection of the
liquid sample within the range of
0.1-100 mL of volume under
high pressure (up to 4000 psi).
 Types of Injector- Manual and
Automated

12.  Variation of temperature during the analytical run can
result in changes of retention time of the separated
eluting components.
 A column oven maintains constant column temperature
using air circulation. This ensures a constant flow rate
of the mobile phase through the column.
 Cooler are used to maintain low temperatures when
heat labile compounds are to be analyzed.

13.  The column is considered the heart of the
chromatograph.
 Columns are usually made of polished stainless steel, are
between 50 and 300 mm long and have an internal
diameter of between 2 and 5 mm.
 They are commonly filled with a stationary phase with a
particle size of 3–10 µm.

14. GUARD COLUMN ANALYTICAL COLUMN
 Usually short guard column is
introduced before the analytical
column to increase the life of analytic
column by removing particulate
matter and contaminants from the
solvent and sample components that
bind irreversibly to the stationary
phase.
 The component of guard column
packing should be closely similar to
that of analytical column.
 Pellicular-Consists spherical
,nonporous , glass or polymer beads
with typical diameter of 30-40 /µm.
 It is the main place where
separation is carried out.
 The majority of liquid
chromatographic column range in
Length 15-250mm.
 Porous-It consist of porous micro
particles having diameter ranging
from 3-10µm
 Substances used in stationary
phase- silica gel

15.  The HPLC detector, located at the end of the column,
detects the analytes as they elute from the
chromatographic column.
 A detector serves to measure the amount of those
molecules so that it can quantitatively analyze the
sample components.
 The detector provides an output to a recorder or
computer that results in the liquid chromatogram.

16.  UV-Visible Detector/Diode array Detectors
 Florescence Detector

17.  Refraction index Detector
 Electric Conductivity Detector

18.  The data system not only controls all the
modules of the HPLC instrument but it also
takes the signal from the detector and uses it
to determine the time of elution(retention
time) of the sample components and the
amount of sample.
 It also shows the result in terms of
chromatogram.

19.  Chromatogram is a graphical display of
results in form of peaks generated as the
separated components pass through the
detector.
 The chromatogram is a two-dimensional
plot with the ordinate axis giving
concentration in terms of detector response
and the abscissa represents the time.
 The detector gives response as a peak
whose height should be ideally dependent
on concentration of the particular
component.
 From the area measurements using simple
arithmetic it is simple to calculate the
concentration of each component as a
percent of the total.

20. Normal
phase HPLC
Reverse
phase HPLC
Size-
exclusion
HPLC
Ion
exchange
HPLC
Types of HPLC

21. Normal phase HPLC
 In this mode, the column packing is polar and the mobile
phase is non-polar.
 Normal phase separations are performed less than 10% of
the time.
 The technique is useful for water-sensitive compounds,
geometric isomers, cis-trans isomers, chiral compounds.

22. Reverse phase HPLC
 The column packing is non-polar and the mobile phase is water (buffer)
+ water-miscible organic solvent.
 Reverse phase separations are performed about 90% of the time.
 The technique can be used for non-polar, polar, ionizable and ionic
molecules.
 The organic solvent increases the solvent strength and elutes
compounds that are very strongly retained on the RPC packing.

23. Size-exclusion HPLC
 In SEC, there is no interaction between the sample
compounds and the column packing material.
 Instead, molecules diffuse into pores of a porous medium.
Depending on their size relative to the pore size, molecules
are separated.
 Molecules larger than the pore opening do not diffuse into
the particles, while molecules smaller than the pore
opening enter the particle and are separated. Large
molecules elute first. Smaller molecules elute later
 The SEC technique is used by 10-15% of
chromatographers, mainly for polymer characterization
and for proteins.

24. Ion exchange HPLC
 In ion exchange, the column packing contains ionic
groups and the mobile phase is an aqueous buffer.
 Ion exchange is used by about 20% of the liquid
chromatographers.
 The technique is well suited for the separation of
inorganic and organic anions and cations in aqueous
solution.
 Ionic dyes, amino acids, and proteins can be separated
by ion exchange.

25. Applications

26. Limitations
 It is difficult to detect co-elution (two compounds
escaping from the tubing at once) with HPLC, which
may lend to inaccurate compound categorization.
 HPLC can also be graded as high priced liquid
chromatography.