1. HAFIZ
MUHAM
MAD
WASEEM
BA BA G
LAHORE
Micrometry

2. Advanced Analytical Techniques
UNIVESITY OF
EDUCATION
LAHORE

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

6. Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Principle

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

11. Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Method

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

15. Advanced Analytical Techniques
Thin layer
chromatography (TLC)-
Applications

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