An Introduction to High
Performance Liquid
Chromatography(HPLC)
Ishpuneet Kaur
M.Sc (1st semester)
3025
Contents
Introduction
Basic Principle
Instrumentation
Instrumentation parameters
Advantages
Limitations
Applications
References
Introduction
HPLC was first developed in 1940 by
Martin and Synge.
The first instrumental liquid
chromatograph was constructed by Csaba
Horvath at Yale University in 1964.
HPLC is used to separate out different
components of mixtures .
Basic Principle
HPLC is a technique in which the
components of a given sample are
separated between two phases i.e.
Stationary phase
Mobile phase
Instrumentation
Solvents must be degassed to
eliminate formation of bubbles. The
pumps provide a steady high
pressure with no pulsating, and can
be programmed to vary the
composition of the solvent during
the course of the separation.
Detectors rely on a change in
refractive index, UV-VIS
absorption, or fluorescence after
excitation with a suitable
wavelength.
Instrumentation
Mobile Phase Reservoir
Solvent Delivery System
Sample Injector port
Column
Detector
Recorder
HPLC
Chromatograms
Absorbance →
Peak A Peak B
0 1 2 3 4 5 6 7
Time (minutes)
Rt = 3.0 min. Rt = 5.2 min.
faster moving slower moving
less retained more retained
Instrumentation Parameters
Internal diameter
A wide range of analytical column
diameters is available, ranging from
4.6mm internal diameter to less than
0.2mm.
Wider the column higher the loading
capacity, hence greater the sample size
that may be injected.
Larger internal diameter columns (over 10 mm) are
used to purify usable amounts of material because of
their large loading capacity.
Analytical scale columns (4.6 mm) have been the most
common type of columns, though smaller columns are
rapidly gaining in popularity. They are used in
traditional quantitative analysis of samples and often
use a UV-Vis absorbance detector.
Narrow-bore columns (1-2 mm) are used for
applications when more sensitivity is desired either with
special UV-vis detectors, fluorescence detectors or with
other detection methods like
liquid chromatography-mass spectrometry
Capillary columns (under 0.3 mm) which are used
almost exclusively with alternative detection means
such as mass spectrometry. They are usually made
from fused silica capillaries.
Particle Size
Most traditional HPLC is performed with
the stationary phase attached to the
outside of small spherical silica particles
(very small beads).
These particles come in a variety of sizes
with 5μm beads being the most common.
Smaller particles generally provide more
surface area and better separations.
Pore Size
Many stationary phases are porous to
provide greater surface area. Small pores
provide greater surface area while larger
pore size has better kinetics especially for
larger analytes. For example a protein
which is only slightly smaller than a pore
might enter the pore but not easily leave
once inside.
Pump Pressure
Pumps vary in pressure capacity, but their
performance is measured on their ability
to yield a consistent and reproducible
flow rate. Pressure may reach as high as
40 MPa, or about 400 atmospheres.
Modern HPLC systems have been
improved to work at much higher
pressures, and therefore be able to use
much smaller particle sizes in the columns
(< 2 micrometers).
Detectors
The function of the detector in HPLC is to
monitor the mobile phase as it emerges
from the column.
Detector can be categories as:
Bulk property detectors
Solute property detectors
ADVANTAGES
Versatile
Stationary supports with very small
particle sizes and large surface areas
Appliance of high pressure to solvent
flow
LIMITATIONS
It is a tentative qualitative, analytical tool
unless coupled with mass spectrometer.
Sample preparation is often required
sample has to load into liquid state.
Only one sample can be analyzed at a
single time.
Resolution may be difficult to attain in
case of complex mixtures.
Applications of HPLC in forensic
science
Drugs: many controlled substances are
analyzed by HPLC. Drugs taken from body
fluids can also be analyzed.
Soils: organic extractions can be done on
soils and various substances separated. The
result is a profile of the soil.
Explosives: it may not be safe to run
explosive extract by GC because of the high
heat but HPLC is an ideal method for
separations of explosives residues.
Inks and dyes: Determination of the Vis
and UV spectra of inks is useful in
comparing a writing instrument to write on
a document. It can also be used in
analyzing the age of ink. Fiber dyes can be
extracted from fibers and separated by
HPLC.
REFERENCES
Thomas M J K (2007): VOGEL’S Text book of
Quantitative Chemical Analysis(6TH Edition),Dorling
Kindersley, India Pvt. Limited.
Lee C Henry(1989):Advances in Forensic Science( Vol
2).Medical Publisher, Chicago.
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