TLC - 25-10-2024

Thin Layer Chromatography (TLC)
Thin Layer Chromatography

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)”
Makes use of:

– A stationary phase: solid
– Mobile phase: Liquid
Basic principle of separation:

– Differential interactions of molecules with these 2 phases
How this works, continued
Molecules that partition into the mobile
phase:
– Will move more rapidly than molecules that partition into the
stationary phase
– Therefore, they will separate from those that are partitioned
into the stationary phase
Faster moving component

partitioned more into Solvent movement
mobile phase
Slower moving component

partitioned more into Two component mixture
stationary phase applied to TLC plate
THIN LAYER CHROMATOGRAPHY
As the mobile phase rises up the

TLC plate by capillary action, the
components dissolve in the solvent
and move up the TLC plate.
http://www.instructables.com/id/EW1YDCYF4REC0IU/
Individual components move up at

different rates, depending on
intermolecular forces between the
component and the silica gel
stationary phase and the component
and the mobile phase.
THIN LAYER CHROMATOGRAPHY
Common stationary phase is SiO2 which is very “polar”.
• It is capable of strong dipole-dipole and H-bond

interactions with the “analytes” (the components being
analyzed).
• More polar analytes interact more strongly with the

stationary phase and move very slowly up the TLC plate.
• More nonpolar analytes interact less strongly with the

polar silica gel and move higher up the TLC plate.
CLASSIFICATION OF ADSORBENTS
USED
1. Classification according to binding strength:
A. Weak adsorbent: sucrose, starch, talc, cellulose
B. Intermediate adsorbent: silica gel, calcium carbonate,
calcium phosphate, magnesia
C. Strong adsorbent: alumina, charcoal
2. Classification according to nature:

A. Inorganic adsorbent: Silica, Silica gel, Alumina, Calcium
phosphate, Glass powder, Kieselguhr ,Magnesium silicate,
Calcium silicate, Phosphate , Ferric & Chromic oxides, Zinc
carbonate & zinc ferro cyanides, Bentonites
B. Organic adsorbent: Normal cellulose powder, Charcoal &

activated carbon, Starch, Sucrose, Manitol, Dextran gel
 SILICA GEL is granular porous form of silica
 Made synthetically from sodium silicate
 Due to silica gel polarity – non polar components tend to
elute before polar ones hence named as Normal-Phase
Chromatography (NPC)
 Hydrophobic groups (C18) attached to silica gel then polar
components elute first hence names as Reverse Phase
Chromatography (RPC).
Polar Non-polar
Comparison of Normal Phase & Reverse Phase :
PARAMETER NORMAL PHASE REVERSE PHASE
Stationary phase Polar Non-polar

Mobile phase Non-polar Polar
Compound eluted Non-polar Polar
first
Compound eluted Polar Non-polar
last
Example of Silica gel C4 ,c8 –bonded
stationary phase phase
CELLULOSE
 Cellulose (C6H10O5)n is a long chain polymeric

polysaccharide carbohydrate of β – glucose
 Adsorbed water or alcohol can be retained by interaction

with hydroxyl groups
 Two types of cellulose are used in planar chromatography:

1.Polymerization b/w 400-500 glucopyranose units
2. 40 – 200 glucopyranose units
ALUMINIUM OXIDE
 It is a chemical compound of aluminum and oxygen with

chemical formula – Al2O3
 Commonly referred to as alumina
 Manufactured in 3 pH ranges – acidic, basic and neutral
 Acidic compounds – phenols, sulphonic, carboxylic &

Amino acids are separated on acidic alumina
 Basic compounds – amines , dyes separated
 Neutral compounds – aldehydes, ketones & lactones

MOBILE PHASE
1) Nature of the substance to be separated i.e whether it is
polar or non-polar.
2) Mode of Chromatography
3) Nature of Stationary phase
4) Mode Separation i.e Analytical or Preparative technique
 Examples: 1) Petroleum ether 2) Cyclohexane

3) Acetone 4) Toluene
5) Ethyl acetate 6) Benzene
7) Alcohols 8) Water
9) Chloroform 10) Pyridine
Rf Values
Molecules that are separated will migrate as “spots” and their
migration can be measured
Migration usually reported as an “Rf” value
Calculation of an Rf value:
– Ratio of sample migration (how far spot moved) to solvent
migration (how far the solvent moved)
Can be used to identify components in a mixture

– Compared to standards
Calculating an Rf Value
Solvent Front
Solvent Front = 5 cm
Spot 1 moved 1 cm
Rf = 1/5 = 0. 2
Origin
Spot 2 moved 4 cm
Spot 1 Spot 2 Rf = 4/5 = 0. 8
Factors which will affect Rf value
1. Type of paper
2. Solvent composition
3. Temperature
4. Chamber saturation
Factors which will NOT affect Rf value
1. Solvent volume
2. Size of paper
3. Sample size
TWO DIMENSIONAL DEVELOPMENT
Sometimes chromatography with a single solvent is not

enough to separate all the constituents of a mixture.
In this case the separation can be improved by two-

dimensional chromatography, where the
chromatography paper is turned through 90° and run a
second time in a second solvent.
Solvent
front Solvent
Paper dried and front
rotated clockwise
through 90o
Mixture of
amino acids
on origin line
Second solvent
First solvent
Two-way chromatography provides better separation of substances that behave
in a similar fashion in the first solvent.
A second run in a different solvent resolves two very close spots more clearly
NEXTX
VISUALIZATION METHOD
 Most of the time spots wont show unless visualized.
 Visualization is a method used to render TLC spots visible
 A visualization method can be:
✓ UV light
✓ Iodine vapors to stain spots
✓ reagents that selectively stain spots leaving others
unaffected
✓ Spraying of chemical indicators: Ninhydrin, H2SO4,
Rubeanic acid, Ammonium sulphide.
✓ Densitometer
Following detecting tech. can also be
categorized as
1) Destructive techniques
Specific spray reagents, samples destroyed before

detection e.g. – ninhydrin reagent
2) Non-destructive techniques
UV chamber, iodine chamber

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Thin Layer Chromatography (TLC)

Thin Layer Chromatography

Makes use of:

Basic principle of separation:

Faster moving component

Slower moving component

As the mobile phase rises up the

Individual components move up at

Common stationary phase is SiO2 which is very “polar”.

• It is capable of strong dipole-dipole and H-bond

• More polar analytes interact more strongly with the

• More nonpolar analytes interact less strongly with the

2. Classification according to nature:

B. Organic adsorbent: Normal cellulose powder, Charcoal &

PARAMETER NORMAL PHASE REVERSE PHASE

Stationary phase Polar Non-polar

 Cellulose (C6H10O5)n is a long chain polymeric

 Adsorbed water or alcohol can be retained by interaction

 Two types of cellulose are used in planar chromatography:

 It is a chemical compound of aluminum and oxygen with

 Commonly referred to as alumina

 Manufactured in 3 pH ranges – acidic, basic and neutral

 Acidic compounds – phenols, sulphonic, carboxylic &

 Basic compounds – amines , dyes separated

 Neutral compounds – aldehydes, ketones & lactones

 Examples: 1) Petroleum ether 2) Cyclohexane

Migration usually reported as an “Rf” value

Can be used to identify components in a mixture

Factors which will NOT affect Rf value

Sometimes chromatography with a single solvent is not

In this case the separation can be improved by two-

 Visualization is a method used to render TLC spots visible

 A visualization method can be:

Specific spray reagents, samples destroyed before

UV chamber, iodine chamber

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