Intro To HPLC system - Lab Training
quantification of constituents of complex organic samples.
At first
sight the assembly of complex modules and bunch of tubings might scare you.
However, on gaining an insight into the role of each component the mystery will
start unfolding and the HPLC system will turn out to be a user-friendly piece
of equipment that if properly looked after will ensure highest accuracy and
reliability of results analysis after analysis.
This is a concise introduction
to the components of HPLC. It is important to understand the individual
component parts and how each contributes to the overall reliability of
analysis. Your increased comfort level will make you realize that HPLC is a
simple to use system and holds promise of unlimited applications.
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.
Different
combinations of these parts namely :
- Pumps
- Detectors
- Injectors yield an infinite
number of configurations based on the application.
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.
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.
What is
HPLC?
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.
Mobile
Phase
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.
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.
Mobile
Phase
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.
Mobile
Phase Reservoirs
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.
see more details in Precautions in the handling of HPLC mobile phase
see more details in Precautions in the handling of HPLC mobile phase
Pumps
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.
Injectors
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.
Column
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.
Column
Oven
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.
See also:Guard columns and their benefits in HPLC analysis - Lab Training
See also:Guard columns and their benefits in HPLC analysis - Lab Training
Detector
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.
Data
Acquisition & Control
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.
Specialized
Versions
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
*lab-training*
No comments