HPTLC means
High Performance Thin Layer Chromatography. It is a modified form of TLC or
Planar Chromatography or flatbed Chromatography as here the Stationary phase
is spread over a support material as a thin layer or flatbed.
In HPTLC
is a method which includes following different stage involved.
Preparation
of Sample:
Proper
sample preparation is an important pre-requisite for the success of TLC separation.
For
normal chromatography: Solvent should be non-polar and volatile.
For
reversed chromatography: Polar solvent is used for dissolving the sample
Sample
and reference substances should be dissolved in the same solvent to ensure
comparable distribution at starting zones.
Selection
of chromatographic plate:
Pre
coated plates are available. In this, the stationary phase is coated over a
solid support like glass, polyester or aluminum. Generally, the thickness
of the sorbent layer is 100-250 μm is used. But for preparative work 1.0 and
2.0 mm are also available.
Prewashing
and Preconditioning:
To avoid
any possible interference due to impurities with the chromatographic separation
particularly in case of quantitative work, it is always recommended to clear
the plate before actual chromatography.
Different
prewashing method Ascending method, a Dipping method, continuous method are used.
Ascending
Method:
In this
technique the chromatographic plates are run blank (i.e. before application of
the sample with a suitable solvent / mobile phase. The solvent/mobile phase
carries the impurities to the top of the plate.
It
takes longer time but the cleaning effect is superior.
It takes
longer time but the cleaning effect is superior.
The
disadvantage of this technique is active dirt gets accumulated at the solvent
front
Dipping
method
In this
technique, the chromatographic plate is dipped in a suitable solvent for
specified period of time, removed from the chamber, and finally dried.
Dipping method is quicker and yields uniform layer but the cleaning effect is often not as
good as the Ascending method.
Continuous method:
In this technique, the
plate to be washed is placed in a chamber having an entrance and exit slits.
The solvent is made to flow
continuously through the chamber that carries the impurities from the plate.
The wanted plates should
always be stored in a dust-free atmosphere, under ambient conditions.
Usually desiccators of
suitable size is used for the storage of plates.
Preconditioning of
Chromatographic:
Chamber saturation has a
pronounced influence on the separation profile.
The time required for the
saturation depends on the mobile phase.
If plates are introduced
into the unsaturated chamber, during the course of development, the solvent
evaporates from the plate mainly at the solvent front and it results in
increased Rf values.
If the tank is saturated prior
to the development, solvent vapors soon get uniformly distributed throughout
the chamber. As soon as the plate is kept in such a saturated chamber, it soon
gets pre-loaded with solvent vapors thus less solvent is required to travel a
particular distance, resulting in lower Rf values.
But in some cases depending
on their polarity saturation and non-saturation of chambers are required:
Eg: Pre-equilibrium is
often recommended in case of solvent with high polarity.
Development in a
non-saturation or partially saturated atmosphere is recommended in solvents
used in a composition leading to phase separation such as a mixture of n-butanol
, water, and glacial acetic acid.
Preloading of dry layer
with solvent, vapors should be avoided for low polar molecules.
Activation of Plate:
Plates are placed in the oven
at 110o-120oc for 30 min prior to the sample application.
Activation at higher
temperature for a longer period is avoided as it may lead to very active layers
and the risk of the samples being decomposed.
Chromatogram development
The different methods used
for development of chambers are like-Ascending, descending .2-dimensional,
horizontal , multiple overrun , gradient ,radial ,anti-radial ,multimodal
, forced flow planar chromatography.
Plates are spotted with
sample and air dried and placed in the developing chambers.
After the development plate
is removed from the chamber and mobile phase is removed under fume cup-board to
avoid contamination of the laboratory atmosphere.
The plates should be always
laid horizontally because when the mobile phase evaporates the separated components
will migrate evenly to the surface where it can be easily detected
Drying of
plate
Drying of
chromatogram should be done in vacuum desiccators with protection from heat and
light.
If hand the dryer is used there may be chances of getting contamination of plates,
evaporation of essential volatile oils if any present in the spot or compounds
sensitive to oxygen may get destroyed due to the rise in temperature.
Detection
of spot Visualization
One of
the characteristic feature of HPTLC is the possibility to utilize
post-chromatographic offline derivatization
Detection is of two types:
• Qualitative
• Quantitative
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