I am HAFIZ M WASEEM FROM mailsi vehari
BSc in science college Multan Pakistan
MSC university of education Lahore Pakistan
i love Pakistan and my teachers
3. Introduction of TLC
It is a technique to separate
components of non-volatile
mixtures using a thin
stationary phase on some
plates (glass)
It may be used for analytical or
preparatory scale
It is performed on a sheet of
glass, plastic or aluminium foil
which is coated with a thin
layer of adsorbent material
like silica gel, aluminium
oxide (alumina) or cellulose
This layer of adsorbent acts as
the stationary phase
4. Introduction of TLC
A mixture is separated on the
basis of affinities for the
mobile and stationary phases
A solvent (mobile phase) draws
up the plate via capillary action
and separates components of a
mixture
Different components rise TLC
plate at different rates which
results in the separation
Rate of upward movement of
the sample on the plate
depends on how tightly the
sample binds with the
stationary phase
5. Introduction of TLC
Spots of the sample on the
plate are visualized by various
methods
Distance travelled by a
component is measured as
compared with the distance
covered by the solvent in a
given time
This is named as retardation
factor (Rf) which ranges from
zero to one
This technique is being widely
used due to its simplicity, low
cost, high sensitivity and fast
speed
7. Principle of TLC
Principle of TLC is similar to
paper chromatography
Different compounds in the
sample travel at different
rates due to different
affinities for the stationary
phase and the solvent
Separation is based on the
competition between the
solute and the mobile phase
for binding places on the
stationary phase
Rf values are used to identify
the compounds because of
their uniqueness
8. Principle of TLC
Rf is the distance travelled by a
component divided by the
distance by the solvent
Rf values depend on polarity of
the components, stationary as
well as mobile phase
More polar compound have
stronger interaction with the
silica (polar stationary phase)
as compared with the less
polar compounds
As a consequence the less polar
compound moves higher up
the plate and have higher Rf
values
9. Principle of TLC
For more polar mobile phase
all compounds on TLC plate
move high up
Generally a substance whose
structure resembles to that of
the stationary phase will have
low Rf value
Rf values depend on layer
thickness, moisture on the TLC
plate, temperature, nature of
mobile phase, nature of TLC
plate, sample size, etc.
Mobile phase must not be
allowed to reach the end of
the stationary phase
10. Principle of TLC
Rf values of the known
compounds or standards are
calculated
Unknown compounds are
identified by comparing with
Rf values of the known ones
Compounds which are more
soluble in the mobile phase
(solvent) will travel high up on
the plate
Similarly compounds more
similar to the stationary phase
will travel less on the plate
than the different ones during
separation
12. Sample application
Different samples are applied in
a row of spots at the same
distance from the bottom edge
Each sample will move in its
own lane from the starting
point
Samples are applied just above
the solvent layer so that they
are not dissolved
A standard is also applied for
the comparison
A co-spot is also applied by
applying the sample on the
standard
Method of TLC
13. Solvent and capillary action
A small amount of solvent is
poured in a container
The container is closed with a
cover glass to achieve the
equilibrium and to avoid the
evaporation
Solvent is used according to
the sample being analyzed
Solvent is allowed to rise up
the plate by capillary action
about one cm below the upper
end of the plate
Solvent is never allowed to
run the end of the plate
Method of TLC
14. Plate visualization
TLC plates are pre-coated with a
fluorescent material such
as zinc sulfide which allows
some of the compounds to be
visualized when examined
under UV light
Plates can also be visualized by
applying other chemicals after
elution
Various visualizing agents react
with the spots to produce
different results
Use of different agents depends
on the types of samples
Method of TLC
16. Measure of sample purity
Purity of sample is analyzed by
direct comparison of the
sample and the standard
In case of impurities extra spots
are detected on visualization of
the plates
Identification of compounds
TLC can be used for purification,
isolation and identification of
the natural products like
alkaloids, steroids, etc.
Standards are used for the
comparison
Applications of TLC
17. Analysis of reactions
Reaction mixture is examined
to assess whether certain
reaction is complete or not
Formation of specific spots
on the plates indicate the
presence of certain products
Biochemical analysis
TLC is useful in separation of
biochemical metabolites from
body fluids like blood plasma,
urine, etc.
Other samples are also used
to identify the metabolites
Applications of TLC
18. Pharmaceutical industry
TLC technique is used for
detection of impurities in the
pharmaceutical products
Various medicines like
sedatives, antihistaminics,
analgesics, local anaesthetics,
may be tested qualitatively by
TLC method
It is also possible to analyze
various intermediate products
formed in the development of
pharmaceutical products
TLC can be applied in studying
kinetics of the products
Applications of TLC
19. Chemical analysis
TLC is used for separation
and identification of various
compounds closely related to
each other
It is also used in the
identification of cations and
anions in inorganic chemistry
Food and cosmetics
TLC method is used for
separation and identification
of colors, preservatives, etc.
It is also used in analysis of
various cosmetic products
Applications of TLC