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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
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
Versatile
Stationary supports with very small
particle sizes and large surface areas
Appliance of high pressure to solvent
flow
LIMITATIONS