C3. TLC

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• INTRODUCTION
• PRINCIPLE
• METHODOLOGY
• ADVANTAGES AND DISADVANTAGES
• APPLICATION
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• Thin layer chromatography (TLC) is an
important technique for identification and
separation of mixtures of organic compounds.
• It is useful in Identification of components of a

mixture (using appropriate standards)
following the course of a reaction, analyzing
fractions collected during purification,
analyzing the purity of a compound.
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• The developing solvent in the mobile phase is the transport medium for the solute to be
separated as it migrates through the stationary phase by capillary forces. The movement
of substances during TLC is the result of two opposing forces, the driving force of the
mobile phase and the retarding action of the adsorbent. The driving force tends to move
the substance from the origin in the direction of the mobile phase flow. The resistive
action impedes the movement of the substance by dragging them out of the flowing
phase back on to the sorbent. Thus the substances adsorbed on the stationary phase.
• The degree of adsorption of a solute on the stationary phase.
K = Cs/Cm where, K- distribution coefficient
Cs- equilibrium concentration of the solute in stationary phase
Cm- equilibrium concentration of the solute in mobile phase
• The distance that solute move up is measured and the Rf (retention factor) value is
calculated for qualitative analysis of the solute.
• Rf= Distance travelled by the solute from the origin/ Distance travelled by the solvent
from the origin
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1. STATIONARY PHASE
• The solid layer supported on a non-porous plate in TLC is generally is called
the adsorbent. Adsorption is the ability of a solid to attract other molecules to
its surface and hold them at the surface. The activity of an adsorbent is
determined by its surface area, its chemical nature and the geometrical
arrangement.
• E.g.
– Weak: Sucrose, Starch, Inulin, Talc, Sodium carbonate
– Medium: Calcium carbonate, Calcium phosphate, Magnesium phosphate,
Calcium hydroxide.
– Strong: Activated magnesium silicate, Activated alumina, Activated
charcoal, Fuller’s earth etc. 5

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3. PREPARATION OF THIN LAYER IN PLATES
The various methods of preparing layers are as follows
: a measured amount of the slurry is put on a given size plate

which is kept on a level surface. The plate is then move back and forth
to spread the slurry uniformly over the surface.
: plates are dipping them at a time, back to back in chloroform

or chloroform-methanol slurries of the adsorbent.
: a small point sprayer was employed for distribution of the

slurry on the glass plate.
: slurry is placed in an applicator. This is either moved over

the stationary and the plate is pushed or pulled through.
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Activation of plates
• After preparation plates are allowed to dry in

air and further dried and activated by heating
at about 110ºC for 30 minutes in hot air oven.
• By removing the liquids associated with layer

completely, the adsorbent layer is activated.
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4. MOBILE PHASE
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5. SPOTTING THE TLC PLATE
One advantage TLC has over other methods is that it is truly a micro scale
technique. Only a few micrograms of sample are necessary to observe the solute
on a TLC plate.
Dissolve a few mg of material in volatile solvent creating a dilute solution
↓
Choose a volatile solvent that completely dissolve the sample
↓
Use a spotting capillary to add the sample to the plate
↓
Use a pencil to spot the solute. This is known as the origin.
• [note: Pencil is always used to mark a TLC plate since the graphite carbon is
inert]
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6.DEVELOPMENT OF CHROMATOGRAM
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Ascending chromatography
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Descending chromatography
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Horizontal development
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7. EVALUATION OF THE CHROMATOGRAM
a. QUALITATIVE: Rf Value method
Because the distance travelled by a substance, relative to the distance travelled by

the solvent front, depends upon the molecular structure of the substance, TLC can
be used to identify substances as well as to separate them. The relationship between
the distance travelled by the solvent front and the substance is usually expressed as
the Rf value:
Rf= distance travelled by solvent front
distance travelled by substance
The Rf values are strongly dependent upon the nature of the adsorbent and solvent.
Therefore, experimental Rf values and literature values do not often agree very well.
In order to determine whether an unknown substance is the same as a substance of
known structure, it is necessary to run the two substances side by side in the same
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chromatogram, preferably at the same concentration.
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b. QUANTITATIVE
Direct method
Indirect method:
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UV VISUALIZATION
IODINE
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1. Purity of sample: more spots other than component to be detected means
impure.
2. Examinations of reactions: whether the reaction complete on not.
3. Check on purification process like distillation, molecular distillation.
4. Identification of compounds like oils, fixed oils, waxes, terpenes, alkaloids,
steroids etc.
5. Biochemical analysis of drugs in body fluids.
6. In chemistry- identification of closely related compounds, cations and
anions
7. Drug monitoring include
Antiepileptic
Analgesics
Abused drugs
Anti-parkinsonism drug
Anti-maniac drugs
NSAID
Drugs acting cardiovascular system
Anti-histamines
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1

• ADVANTAGES AND DISADVANTAGES

• It is useful in Identification of components of a

• The solid layer supported on a non-porous plate in TLC is generally is called

the adsorbent. Adsorption is the ability of a solid to attract other molecules to

– Weak: Sucrose, Starch, Inulin, Talc, Sodium carbonate

– Medium: Calcium carbonate, Calcium phosphate, Magnesium phosphate,

– Strong: Activated magnesium silicate, Activated alumina, Activated

charcoal, Fuller’s earth etc. 5

The various methods of preparing layers are as follows

: a measured amount of the slurry is put on a given size plate

: plates are dipping them at a time, back to back in chloroform

: a small point sprayer was employed for distribution of the

: slurry is placed in an applicator. This is either moved over

• After preparation plates are allowed to dry in

• By removing the liquids associated with layer

Because the distance travelled by a substance, relative to the distance travelled by

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