Simplify Grasp of HPLC by Relating it to Human Body Functions
Chromatographic
analysis has made innumerable contributions in research and quality control
laboratories. The versatility and acceptance of the chromatographic techniques
is showing an outstanding acceptance in sectors covering fundamental and
applied research and in industrial sectors covering quality control in
pharmaceuticals, foods, environmental monitoring, clinical diagnostics
monitoring, forensic samples analysis, etc.
Applications
involving HPLC analysis have seen remarkable growth in recent years. This has
been possible because of availability of options in choice of separation columns,
detectors, scale of analysis (pg to gm. level determinations) and modes of
operation such as reverse phase, normal phase, ion chromatography and gel
permeation chromatography.
HPLC
is a modular system and the reliability of results
depends greatly on the performance of each and every module. In a way
similarities can be drawn with the functioning of organs of the human body. We
shall see the comparison in the next few sections.
Mobile Phase- the Blood lifeline
In
the human system the blood stream serves transportation of the nutrients and
oxygen to each and every organ to promote healthy function and growth of cells.
Likewise the mobile phase serves the purpose of transferring the injected
sample through the HPLC system- first through the separation column and next to
the detector. Like the blood stream the mobile phase flows through the tubes
and column inside the HPLC system in a defined path. The reproducibility of results
depends on the pre-defined flow rate without any restrictions or leakages. The
mobile phase should be prepared fresh before start of analysis, degassed to
free it from dissolved gases and filtered. Filtration under vacuum of the
mobile phase helps remove any solid suspensions which can result in blockages
and damage delicate components of the system.
Sample- The food that you eat
The
sample should be fully soluble in the mobile phase and filtered to remove any
solid suspensions. This is comparable to food which should be chewed properly
for easy and thorough digestion. The sample needs to be fully compatible with
the mobile phase solvents and the column packing material and not lead to any
side reactions. The amount of sample injected should not exceed the recommended
amount otherwise it can lead to overloading of the column and lead to
flat-topped or broad peaks. This can be considered to be akin to indigestion
when food is taken in excess of stomach’s processing capacity.
Column-The Stomach
Stomach
is the key organ for assimilation and digestion of foods. Likewise in HPLC the
column facilitates interaction of sample with the packed stationary phase. The
sample components get attached to the stationary phase and subsequently eluted
in a sequence. The elution rate is temperature dependent so it is essential to
place the column inside a thermos tatted column oven for stabilization and
maintenance of constant column temperature.
Pump- The Heart
Heart
is the principal organ responsible for pumping blood throughout the body in
continuous and consistent flow rate throughout your life-time. Similarly, the
HPLC pump or set of pumps are required to deliver the mobile phase at set or
variable rates depending on the mobile phase delivery program. The pump
requires regular inspection and maintenance to replace worn-out components such
as pistons, seals and O-rings. This is necessary to prevent leaks in the systems
which affect the flow rates.
Detector-Eyes and Brain
Eyes
and brain help us identify the objects that we see and transmit the signals to
the brain to help determine the number of objects or to estimate the quantity
of material. Similarly the detector identifies the individual separated
components separated by the column and produce electrical signals which help
the detector quantify them through the application software. The detector
should be free of any leftover components of previous analysis before the start
of fresh analysis.
The
objective of comparing the human body to the HPLC system was to make its
understanding much more easy and interesting so that you do not get scared by
the sight of complex tubings, wires and different modules once you come across
such systems and are able to relate it to your body organs for an easy
understanding of its operation.
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