Introduction to High Performance Liquid Chromatography (HPLC)

Introduction to High Performance
Liquid Chromatography and its parts
1

Chromatography equipment looks
rather intimidating to anyone who has
not handled it before, but on a closer look and as
you get familiar with the equipment you realize
that behind the network of wires, complex
plumbing and circuitry is a simple machine with
only a few major parts.
2

Different combinations of these parts namely :
• Pumps
• Detectors
• Injectors
yield an infinite number of configurations based
on the application.
3

Just like an understanding of human anatomy
makes you conscious of the vital role of each
and every body organ towards your well being
and vitality. Similarly you need to have a good
understanding of the parts of your HPLC
system to generate data of highest reliability.
4

A conceptual understanding of the function of
each component will add to your comfort level
with your HPLC system. You will ensure long
time usage with high reliance on output data.
5

HPLC is a technique for separation, identification
and quantification of components in a
mixture. It is especially suitable for
compounds which are not easily volatalised,
thermally unstable and have high molecular
weights.
8

The liquid phase is pumped at a constant rate to
the column packed with the stationary phase.
Before entering the column the analysis
sample is injected into the carrier stream.
10

On reaching the column the sample components
are selectively retained on the basis of
physico-chemical interactions between the
analyte molecules and the stationary phase.
11

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.
14

These are inert containers for mobile phase storage
and transport. Generally transparent glass bottles
are used so that so as to facilitate visual
inspection of mobile phase level inside the
container. Stainless steel particulate filters are
provided inside for removal of particulate
impurities in the mobile phase if any.
17

Variations in flow rates of the mobile phase
effect elution time of sample components and
result in errors. Pumps provide constant flow
of mobile phase to the column under constant
pressure.
20

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.
23

A column is a stainless steel tube packed with
stationary phase. It is a vital component and
should be maintained properly as per supplier
instructions for getting reproducibility and
separation efficiency run after run.
26

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
29

A detector gives specific response for the components
separated by the column and also provides the
required sensitivity. It has to be independent of any
changes in mobile phase composition. Majority of the
applications require UV-VIS detection though detectors
based on other detection technique are also popular
these days.
32

Data Acquisition & Control
33

Modern HPLC systems are computer based and
software controls operational parameters such
as mobile phase composition, temperature,
flow rate, injection volume and sequence and
also acquisition and treatment of output.
34

Specialized HPLC Systems might also have solvent
selection valves, vacuum degasser, auto
samplers, column switches, pre or post
column derivatization and fraction collectors.
These are all covered at length in our online
certificate program on HPLC.
Certificate Course on HPLC
36

What Next?
• Keep Learning to Keep Succeeding – visit
www.lab-training.com/hplc to learn more about
HPLC
• Get online certificate training on HPLC –
www.lab-training.com/product/join-our-certificate-
course-on-hplc/
• Subscribe to our newsletter and get free training
delivered straight to your inbox – www.lab-training.
com
• Don’t have a HPLC? Need samples to be tested
visit www.testing-lab.com and get a proposal!
37

Introduction to High Performance Liquid Chromatography (HPLC)

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