An Introduction to High

Performance Liquid
Performance Liquid
Chromatography(HPLC)
Contents
Introduction
Introduction
Basic Principle
Basic Principle
Instrumentation
Instrumentation
Instrumentation parameters
Instrumentation parameters
Advantages
Advantages
Limitations
Limitations
Applications
Applications
References
References
Introduction
HPLC was first developed in 1940 by
HPLC was first developed in 1940 by
Martin and Synge.
Martin and Synge.
The first instrumental liquid
The first instrumental liquid
chromatograph was constructed by Csaba
chromatograph was constructed by Csaba
Horvath at Yale University in 1964.
Horvath at Yale University in 1964.
HPLC is used to separate out different
HPLC is used to separate out different
components of mixtures .
components of mixtures
Basic Principle
HPLC is a technique in which the
HPLC is a technique in which the
components
of a given sample are
components
of a given sample are
separated between two phases i.e.
separated between two phases i.e.
Stationary phase
Stationary phase
Mobile phase
Mobile phase
Instrumentation
Solvents
Solvents must be degassed to
must be degassed to
eliminate formation of bubbles. The
eliminate formation of bubbles. The
pumps provide a steady
pumps provide a steadyhigh
high
pressure
pressure with no pulsating, and can
with no pulsating, and can
be programmed to vary the
be programmed to vary the
composition of the solvent during
composition of the solvent during
the course of the separation.
the course of the separation.
Detectors
Detectors rely on a change in
rely on a change in
refractive index
refractive index,
,
UV-VIS
UV-VIS
absorption
absorption, or
, orfluorescence
fluorescenceafter
after
excitation with a suitable
excitation with a suitable
wavelength.
wavelength
Instrumentation
Mobile Phase Reservoir
Mobile Phase Reservoir
Solvent Delivery System
Solvent Delivery System
Sample Injector port
Sample Injector port
Column
Column
Detector
Detector
Recorder
Recorder
HPLC
Chromatograms

Instrumentation Parameters
Internal diameter
Internal diameter
A wide range of analytical column
A wide range of analytical column
diameters is available, ranging from
diameters is available, ranging from
4.6mm internal diameter to less than
4.6mm internal diameter to less than
0.2mm.
0.2mm.
Wider the column higher the loading
Wider the column higher the loading
capacity, hence greater the sample size
capacity, hence greater the sample size
that may be injected.
that may be injected
Larger internal diameter columns (over 10 mm) are
used to purify usable amounts of material because of
used to purify usable amounts of material because of
their large loading capacity.
their large loading capacity.
Analytical scale columns (4.6 mm) have been the most
Analytical scale columns (4.6 mm) have been the most
common type of columns, though smaller columns are
common type of columns, though smaller columns are
rapidly gaining in popularity. They are used in
rapidly gaining in popularity. They are used in
traditional quantitative analysis of samples and often
traditional quantitative analysis of samples and often
use a
use a UV-Vis absorbance detector
UV-Vis absorbance detector.
.
Narrow-bore columns (1-2 mm) are used for
Narrow-bore columns (1-2 mm) are used for
applications when more sensitivity is desired either with
applications when more sensitivity is desired either with
special UV-vis detectors,
special UV-vis detectors,fluorescence
fluorescence detectors or with
detectors or with
other
detection
methods
like
other
detection
methods
like
liquid chromatography-mass spectrometry
liquid chromatography-mass spectrometry
Capillary columns (under 0.3 mm) which are used
Capillary columns (under 0.3 mm) which are used
almost exclusively with alternative detection means
almost exclusively with alternative detection means
such as
such asmass spectrometry
mass spectrometry. They are usually made
. They are usually made
from
from fused silica
fused silicacapill aries.
capillaries.
Particle Size
Most traditional HPLC is performed with
Most traditional HPLC is performed with
the stationary phase attached to the
the stationary phase attached to the
outside of small spherical silica particles
outside of small spherical silica particles
(very small beads).
(very small beads).
These particles come in a variety of sizes
These particles come in a variety of sizes
with 5μm beads being the most common.
with 5μm beads being the most common.
Smaller particles generally provide more
Smaller particles generally provide more
surface area and better separations.
surface area and better separations
Pore Size
Many stationary phases are porous to
Many stationary phases are porous to
provide greater surface area. Small pores
provide greater surface area. Small pores
provide greater surface area while larger
provide greater surface area while larger
pore size has better kinetics especially for
pore size has better kinetics especially for
larger analytes. For example a protein
larger analytes. For example a protein
which is only slightly smaller than a pore
which is only slightly smaller than a pore
might enter the pore but not easily leave
might enter the pore but not easily leave
once inside.
once inside.
Pump Pressure
Pumps vary in pressure capacity, but their
Pumps vary in pressure capacity, but their
performance is measured on their ability
performance is measured on their ability
to yield a consistent and reproducible
to yield a consistent and reproducible
flow rate. Pressure may reach as high as
flow rate. Pressure may reach as high as
40 MPa
40 MPa, or about
, or about 400 atmospheres
400 atmospheres.
.
Modern
HPLC
systems
have
been
Modern
HPLC
systems
have
been
improved to work at much higher
improved to work at much higher
pressures, and therefore be able to use
pressures, and therefore be able to use
much smaller particle sizes in the columns
much smaller particle sizes in the columns
(< 2 micrometers).
(< 2 micrometers)
Detectors
 The function of the detector in HPLC is to
The function of the detector in HPLC is to
monitor the mobile phase as it emerges
monitor the mobile phase as it emerges
from the column.
from the column.
Detector can be categories as:
Detector can be categories as:
 Bulk property detectors
Bulk property detectors
 Solute property detectors
Solute property detectors
ADVANTAGES
Versatile
Versatile
Stationary supports with very small
Stationary supports with very small
particle sizes and large surface areas
particle sizes and large surface areas
Appliance of high pressure to solvent
Appliance of high pressure to solvent
flow
flow
LIMITATIONS
It is a tentative qualitative, analytical tool
It is a tentative qualitative, analytical tool
unless coupled with mass spectrometer.
unless coupled with mass spectrometer.
Sample preparation is often required
Sample preparation is often required
sample has to load into liquid state.
sample has to load into liquid state.
Only one sample can be analyzed at a
Only one sample can be analyzed at a
single time.
single time.
Resolution may be difficult to attain in
Resolution may be difficult to attain in
case of complex mixtures.
case of complex mixtures.
Applications of HPLC in forensic
science
science
Drugs
Drugs: many controlled substances are
: many controlled substances are
analyzed by HPLC. Drugs taken from body
analyzed by HPLC. Drugs taken from body
fluids can also be analyzed.
fluids can also be analyzed.
Soils
Soils: organic extractions can be done on
: organic extractions can be done on
soils and various substances separated. The
soils and various substances separated. The
result is a profile of the soil.
result is a profile of the soil.

Explosives
Explosives: it may not be safe to run
: it may not be safe to run
explosive extract by GC because of the high
explosive extract by GC because of the high
heat but HPLC is an ideal method for
heat but HPLC is an ideal method for
separations of explosives residues
separations of explosives residues.
.
Inks and dyes:
: Determination of the Vis
Determination of the Vis
and UV spectra of inks is useful in
and UV spectra of inks is useful in
comparing a writing instrument to write on
comparing a writing instrument to write on
a document. It can also be used in
a document. It can also be used in
analyzing the age of ink. Fiber dyes can be
analyzing the age of ink. Fiber dyes can be
extracted from fibers and separated by
extracted from fibers and separated by
HPLC.
HPLC
REFERENCES
Thomas M J K (2007): VOGEL’S Text book of
Thomas M J K (2007): VOGEL’S Text book of
Quantitative Chemical Analysis(6
Quantitative Chemical Analysis(6TH
THEdition),Dorling
Edition),Dorling
Kindersley, India Pvt. Limited.
Kindersley, India Pvt. Limited.
Lee C Henry(1989):Advances in Forensic Science( Vol
Lee C Henry(1989):Advances in Forensic Science( Vol
2).Medical Publisher, Chicago.
2).Medical Publisher, Chicago.
http://
htt
p://www.chemguide.co.uk/analysis/chro
www.chem
guide.co.uk/analysis/chro
matography/hplc
mato

graphy/hplc
http://keats.admin.virginia.edu/ralphs_chemistry_cours
http://keats.admin.virginia.edu/ralphs_chemistry_cours
es/470/9/sld039.html
es/470/9/sld039.html
http://www.chemistry.nmsu.edu/Instrumentation/HPLC_Inst
http://www.chemistry.nmsu.edu/Instrumentation/HPLC_Inst
THANKS