THIN LAYER

CHROMATOGRAPHY
(TLC)
by
Mr. Shaise Jacob
Faculty, Nirmala College of Pharmacy
Muvattupuzha
Kerala, India
 Chromatography
• There are two basic types of
chromatography
– Gas
– Liquid
• Liquid includes TLC and high
performance liquid chromatography
(HPLC)
 Introduction
• TLC is a form of liquid chromatography
consisting of:
– A mobile phase (developing solvent) and
– A stationary phase (a plate or strip coated with a
form of silica gel)
– Analysis is performed on a flat surface under
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
 Normal Phase
• Normal phase is the terminology used 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 than the stationary phase.
 THIN LAYER
CHROMATOGRAPHY
Chromatography is used to separate
mixtures of substances into their
components.
Similar to P.C, except that a thin layer of
some inert material, i.e. Aluminium oxide,
mag.oxid. , sili.oxide is used instead of paper.
• A layer of any one of these oxide is made
from a slurry of power in a suitable inert
solvent.
• Slurry is spread over a flat surface ( glass,
metal or rigid plastic ) & dried
 PRINCIPLE
ADSORPTION
The component with more affinity towards the S.P travels
slower
The component with lesser affinity towards the S.P travels
faster
ADVANTAGES OF TLC
• simple mtd. & cost of the equipment is low
• rapid technique & not time consuming like C.C
• separation of µg of the substances can be achieved
• any type of compound can be analyzed
• corrosive spray reagents can be used without
damaging the plate & needs less solvent
 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 (1)

– 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
 PRACTICAL REQUIREMENTS
1. 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)
Coater, hand operated
 2. GLASS PLATE
Specific dimensions-
20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm
Microscopic slides can also be used
Plates should be of good quality & withstand high
temperatures
3. PREPARATION & ACTIVATION OF TLC
PLATES
♦ Pouring ( simplest 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 100˚C for 30 min.
Then plates may be kept in desiccators
4. APPLICATION OF SAMPLE
» Using capillary tube or micropipette
» Spotting area should not be immersed in the mobile
phase
5. 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”
 6. MOBILE PHASE
M.P used depends upon various factors
► Nature of the substance
► Nature of the S.P
►Mode of Chromatography
►Separation to be achieved,
Analytical/Preparative
e.g. → pyridine, pet. ether, carbon tetrachloride,
acetone, water, glycerol, ethanol, benzene….
 7. DEVELOPMENT TECHNIQUE
1. One dimensional development
2. Two dimensional development
3. Horizontal development
4. Multiple development
8. DETECTING OR VISUALISING AGENTS
a) 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
 Same as P.C
QUALITATIVE ANALYSIS
1. Rx value – The ratio of distance traveled by
the sample & the distance traveled by the
standard.
2. Rf value -
QUANTITATIVE ANALYSIS
> Direct & Indirect method
 APPLICATIONS OF TLC

»Purity of sample
»Examination of reaction
»Identification of compounds
»Biochemical analysis
»In pharmaceutical industry
»Separation of multicomponent pharmaceutical
formulations
»In food and cosmetic industry
Thank you