refrence: biophysics & molecular biology by pranav kumar wilson & walker : instrumentation & biotechniques biology discussion

1. Presented By:-
Shweta Gupta &
Neha Gautam
THIN LAYER
CHROMATOGRAPHY(TLC)

2.  History
 Basic introduction
 Principle
 Components
 Method
 Advantages
 Disadvantages
 Applications

4.  Chromatography is a technique for separating mixtures into their
components in order to analyze, purify, and quantify the
component.
 All the forms of chromatography work on same principle.
 They all have a stationary phase ( a solid, or a liquid supported on
a solid) and a mobile phase( a liquid or a gas).
 The mobile phase flows through the stationary phase that carries
the component of the mixture with it, different component travel
at different rate.
Chromatograph
y
Planer
Paper
Thin layer
Column

5.  Thin layer chromatography (TLC) is a technique
used to identify the components/compounds present
in a mixture by separating them using a thin
stationary phase (silica gel) supported on an inert
substrate and a mobile phase (solvent).
To study the purity of the compound
To study the progress of a reaction
To identify the various components present in the
mixture

6.  Stationary phase- silica gel, alumina etc. over glass metal,
plate, plastic sheet etc
 Silica gel-acidic-steroids, amino acids, hydrocarbons etc.
 Alumina-basic-amines, bile, lipids etc.
 Cellulose-neutral-carbohydrates, sugars etc
 Mobile phase - developing solvent/mixtures of
solvents(reasonably volatile)
 Sample-sample containing mixture of compounds and/or
individual compounds

9.  Developing- developing solvent should be
below the level of spotted samples.
 Mixtures of strongly polar compounds-ethyl
: acetate: butanol : acetic acid:
water(80:10:5:5)
 Polar compounds-10% methanol in DCM
 Strongly basic compounds-10%NH4OH in
methanol in dichloromethane

10. Visualization:
 Colored samples- just dry to vaporize the solvent
 Uncolored samples
 Iodine chamber- place iodine crystals in the chamber
 Fluorescent substance added to stationary phase(silica
gel), which will fluoresce/glow in UV light(254nm)
leaving the dark spots.

11. 1 -TLC PLATES:-
These are stable and
chemically inert
plates, where a thin
layer of stationary
phase is applied on
its whole surface
layer.
The stationary
phase on the plates
is of uniform
thickness and is in
fine particle size.
2-TLC CHAMBER:-
This is used for the
development of the
TLC plate.
The chamber
maintains a stable
environment inside
for proper
development of
spots.
It also prevents the
evaporation of
solvents and keeps the
process dust-free.
3- MOBILE PHASE:-
The mobile phase
used should be
particulate-free and
of the highest
purity for proper
development of TLC
spots.
The solvents
recommended are
chemically inert
with the sample, a
stationary phase.
4-A FILTER PAPER:-
This is moistened in
the mobile phase,
to be placed inside
the chamber.
This helps develop
a uniform rise in a
mobile phase over
the length of the
stationary phase.

12. FIG: TLC SETUP

17. FIG: A- Distribution of spots after migration,
B- Sample spots seen in UV transilluminator

18. Separation of multiple mixtures

19. The rate of migration of the various substances being separated are
governed by their relative solubilities in the polar stationary phase & the
non polar mobile phase.
 During the separation process , a given solute is distributed between
the mobile & stationary phases according to their partition coefficient
(Kd).
Kd= Concentration of solute in phase A
Concentration of solute in phase B
The migration rate of a substance during TLC is usually expressed as
the dimension term Rf (relative front),which is the ratio of the distance
traveled by substances and solvent front.
Rf= Distance traveled by substance
Distance traveled by solvent front

20. FIG: Example of calculating relative
front

21.  It is a simple process with short development time.
 It helps with the visualization of separated compound spots easily.
 The method helps to identify the individual compounds.
 It helps in isolating of most of the compounds.
 The separation process is faster and the selectivity for compounds is
higher (even small differences in chemistry is enough for clear
separation).
 The purity standards of the given sample can be assessed easily.
 It is a cheaper chromatographic technique.

22.  Stationary phase is short
 High detection limit
 Open system – temperature and humidity can
affect the results

23. Test the purity of the
sample
TLC helps to detect the
purity of the sample by
direct comparison with
the standard or
authentic sample. Any
impurity in the sample
shows up as extra spots
in chromatography.
Identify the
components
TLC can purify, isolate
and identify the natural
products like volatile oil
or essential oil, fixed
oil, waxes, terpenes,
alkaloids, glycosides,
steroids etc. in the test
samples.
Biochemical
analysis
Biochemical
metabolites from the
body fluids, blood
plasma, serum, urine
etc. can be isolated
using thin layer
chromatography.
In chemistry
TLC is used to
separate and identify
closely related
compounds or
cations and anions in
inorganic chemistry.

24. Pharmaceutical industries
utilize TLC technique for
qualitative analysis or
detect impurities in various
medicines like hypnotics,
sedatives, anticonvulsants,
tranquillizers, anti-histamines,
analgesics, local anesthetics,
steroids, etc. Yet another
important application of TLC is
to separate multi-
component pharmaceutical
formulations into its individual
components.
In food and cosmetic
industry
Any artificial color,
preservatives,
sweetening agent, and
other impurities in food
and cosmetic products
can be detected and
isolated by TLC
technique.

27. https://youtu.be/yoYECMP_1iI