Guided by : Dr. N.

Ganesh Sir
Presented by : AMBIKESH SONI
 INTRODUCTION

 Chromatography is a physical method of seperation in which the

components to be separated are distributed between the two phases , one
of which is stationary (stationary phase) while the other (the mobile
phase) moves in definite direction.
 There are two basic types of chromatography known :-
A. Gas
B. Liquid
 Liquid includes the TLC (Thin layer chromatography) and HPLC (High
performance liquid chromatography).
TLC is a form of liquid chromatography consisting of
(i) A mobile phase (developing solvent) and ;
(ii) A stationary phase (a plate or strip coated with a
form of silica gel)
(iii) Analysis is performed on a flat surface with atmospheric
pressure and room temperature.
 Michael Tswett is credited as being the father of liquid
chromatography.
 Tswett developed his ideas in the early 1900’s.
 TLC

The two most common classes of TLC are :-

- Normal phase.
- Reversed phase.
Thin layer Chromatography can be defined as a method of
separation or identification of a mixture of components into
individual components by using finely divided adsorbent
solid/ liquid spread over a glass plate and a liquid as a
mobile phase.
 Normal Phase

Normal phase is the terminology use when the

stationary phase is polar ; for example silica gel ,
and the mobile phase is an organic solvent or a
mixture of organic solvents which is less polar
than the stationary phase.
 Reversed Phase

Reversed phase is the terminology used when

the stationary phase is a silica bonded with an
organic substrate such as a long chain aliphatic
acid like C-18 and the mobile phase is a mixture
of water and organic solvent which is more polar
chain the stationary phase.
 THIN LAYER CHROMATOGRAPHY

 Similar to paper chromatography, except that a thin layer of some inert

material ,i.e. Aluminium oxide, magnesium oxide, silicon oxide is used
instead of paper.
 A layer of any one of these oxide is made from a slurry of powder in a
suitable inert solvent.
 Slurry is spread over a flat surface (glass , metal or rigid plastic ) & dried.
 TLC is based on the principle of adsorption i.e the component with more
affinity towards the stationary phase travels slower then the component
with lesser affinity towards the stationary phase travels faster.
 Steps in TLC Analysis

The following are the important components of a typical TLC

system :
- Apparatus (developing chamber)
- Stationary phase layer and mobile phase
- Application of sample
- Development of the plate
- Detection of analyte
 General Procedure

 Decide if you are going to do Normal or Reversed phase chromatography.

 Prepare a plate or select a plate with the proper sorbent material.
 Prepare the mobile phase
 Mark the plate
 Apply the sample
 Develop the plate
 Detect the analytes
 REQUIREMENTS TO PERFORM TLC
STATIONARY PHASE
Adsorbents mixed with water or other solvents
Slurry
Silica gel H ( Silica gel with out binder )
Silica gel G ( Silica gel + CaSO4 )
Silica GF ( Silica gel + binder + fluorescent indicator )
Alumina, Cellulose powder, Kieselguhr G ( Diatomaceous earth +
binder )
 GLASS PLATE
Specific dimension-

#. 20cmx20cm, 20cmx10cm, 20cmx5cm

#. Microscopic slides can also be used
#. Plates should be of good quality &
withstand high temperatures
 PREPERATION & ACTIVATION OF TLC PLATES

#. Pouring (simple methods)

#. Dipping (used for small plates)
#. Spraying (difficult to get uniform
layers)
#. Spreading (best technique)…TLC
Spreader
 ACTIVATION OF PLATES

After spreading Air dry (5 to 10

minutes).
Activated by heating at about 100oC for 30
min.
Then plates may be kept in desiccators.
 APPLICATION OF SAMPLE
 Using capillary tube or micropipette.
 Spotting area should not be immersed in the middle
phase.

DEVELOPMENT TANK
 Better to develop in glass beakers , jars to avoid more
wastage of solvents.
 When standard method is used, use twin trough tanks.
 Do chamber saturation to avoid “edge effect”.
 MOBILE PHASE (M.P.)
M.P. used depends upon various factors :-

 Nature of the substance

 Nature of the stationary phase
 Mode of Chromatography
 Separation to be achieved , Analytical/Preparative.
Eg. pyridine, petroleum ether, carbon tetrachloride, acetone,
water, glycerol, ethanol, benzene........
 DEVELOPMENT TECHNIQUE

 One dimensional development

 Two dimensional development
 Horizontal development
 Multiple development
 DETECTING OR VISUALISING AGENTS
 Non specific methods :-
#. Iodine chamber method.
#. Sulphuric acid spray reagent.
#. UV chamber for fluorescent compounds.
#. Using fluorescent stationary phase.
 SPECIFIC METHODS
 Spray reagents or Detecting agents or Visualizing agents.
#. Ninhydrin reagent.
#. Sulfuric acid
#. Nitric acid
QUANTITATIVE ANALYSIS
 Direct & Indirect method.
QUALITATIVE ANALYSIS
 APPLICATION OF TLC

 Examination of reaction.
 Identification of compounds.
 Biochemical analysis.
 In pharmaceutical analysis.
 In food and cosmetic industry.
 Purity of sample.
EXPERIMENT TO DEMONSTRATE TLC FROM THE LEAVES
OF Anona muricata