Thin layer chromatography (TLC) separates substances based on differential adsorption on inert solids using a mobile phase of solvent. The technique employs various adsorbents for fractionating mixtures, and different methods are used for applying samples and detecting components. TLC is utilized for purity checking, identifying compounds, and monitoring chemical reactions.

1. THIN LAYER
CHROMATOGRAPHY

2.  INTRODUCTION
 PRINCIPLE
 EXPERIMENTAL TECHNIQUE
 EVALUATION OF CHROMATOGRAM
 APPLICATION
 REFERENCES

3.  Thin Layer Chromatography: “The separation of
moderately volatile or nonvolatile substances based
upon differential adsorption on an inert solid
(stationary phase) immersed in a organic solvent or
solvent mixture (mobile phase)”
 Thin layer chromatography was first introduced by
IZMAILOV & SHRAIBER in 1938.

4. Principle of TLC
Adsorption Partition Ion Exchange
Reversed
Phase partition

5.  Chromatography on adsorbents such as silica
gel and alumina has widely been employed for
the fractionation of mixture of non polar
material into class of compounds that migrate
at different rate by virtue of their respective
polarities.
 Eg-
Hydrocarbons are easily separated from
acids,alcohols & aldehydes.

6.  Separation of mixture is effected by virtue of
difference in the solubilities of its components
in developing solvent and the stationary liquid
phase .
Ion Exchange:
 In ion exchange system the rate of migration
of a compound can be found by the total
charge of ionised group per molicule.

7.  The stationary phase is non polar and the
mobile phase is polar .
 R P partition has widely use for fractionation
of hydrocorbons and other non polar
petroleum products.

8. Coating materials
Preparation of thin layer in plates
Activation of adsorbents
Sample Application
Development Tank
Solvent system
Plate Development
Detection of Component
Evaluation of chromatogram

9.  A large no. of coating materials are used
which are commercially produced as a thin
adsorbents.
Adsorbents Acidic or Basic Components to be
separated
Silica gel Acidic Acidic and neutral
substances
Alumina Basic Basic and neutral
Cellulose powder Neutral Water soluble compounds

10.  Methods:
Pouring
Dipping
SprayingSpreading
Precoated
plates

11.  After making thin layer in plates to remove as
completely as possible the liquid associated
with the thin layer.
 This is done by drying the thin layer plate for
30 min in air and then place in oven at 110 C
for 30 min.

12.  Agla microsyring is generally used for
transferring the sample solution on thin layer .

13.  In TLC the plate is placed in a development
chamber at an angle of 45 .
 The bottom of chamber is covered up to 1 mm
by the solvent.

14. Trial & Error:
If one does not know about the
nature of component of mix to be separated the best
elute is found by trial & error method.

15.  If one knows the chemical nature of solute to
be separated it is possible to know a solvent
by using Stahl’s triangle.
hydrophilic
M
non polar
E Lipophilic
polar Inactive
active S

16.  The plate is placed in the closed container
saturated with developing solvent.

17.  Most of methods are used for detection of
separated salute on TLC.
 One can see coloured compound usually.
 One can detect colourless compound by UV
light or with treatment of visualising agent.
Eg.
Iodine vapour
Bromocresol green
Ninhydrine
Dragondroff reagent

18. Methods
Qualitative Quantitative
Direct Indirect

19.  This is done by the help of measurement of
migration parameters.
Migration
parameters
Rf Rx RM

20. solvent front
component B
component A
origin
dS
dB
dA
Rf of component A =
dA
dS
Rf of component B =
dB
dS
The Rf value is a decimal fraction,
generally only reported to two decimal
places

21. Distance travel by substance from origin line
Rx =
Distance travel by std sub x from the origin line
RM = log(1/Rf -1)

22. Direct method:
Visual
assesment of
chromatogram
Measurment of
spot area
Spectrophotometry
Dencitometry

23. Methods
Gravimetric
UV spectroscopy
Fluorimetry
Colourimetry
Calorimetry
Flame photometry

24.  For checking the purity of sample.
 Examination of reactions.
 Identification of organic compounds.
 Separation of amino acids.
 Separation of inorganic ions.
 Progress of chemical reaction .
 As a purification process.

25. 1. R.A.Day, Jr. A.L.Undewood(1987). Analisis
Kualititatif. Edisi ke Empat, ms: 474-529.
2. David G. Watson(2005). Pharmaceutical
analysis. Edisi ke-2, ms 315-331
3. http//orgchem.colorado.edu/hndbooksupport/
TLC/TLCprocedure.html
4. Chatewal R.G.Instrumentatal methods of
chemical analysis,fifth edition 2002
Himalaya publishing house pg no. 2.599