Thin Layer Chromatography

By
Dr. Gurjeet Kaur
Introduction
Thin-layer chromatography or TLC, is a form of chromatography where
the stationary phase is normally a polar absorbent and the mobile
phase can be a single solvent or combination of solvents.
TLC is a quick, inexpensive microscale technique that can be used to:
• determine the number of components in a mixture
• verify a substance’s identity
• monitor the progress of a reaction
• determine appropriate conditions for column chromatography
• analyze the fractions obtained from column chromatography
TLC works on the principle-
The partition coefficient, k is the equilibrium constant for the
distribution of molecules between the mobile phase and the stationary
phase.
Different inks and dyes, depending on their molecular structures and
interactions allow a quick and efficient separation.
In thin-layer chromatography, the stationary phase is a polar absorbent,
usually alumina or silica. This absorbent is coated on a glass slide or
plastic sheet creating a thin layer of the particular stationary phase.
Almost all mixtures of solvents can be used as the mobile phase. By
manipulating the mobile phase, organic compounds can be separated.
Theory
All forms of chromatography involve a dynamic and rapid equilibrium of
molecules between the two phases. There are
1. free - completely dissolved in the liquid or gaseous mobile phase
2. absorbed - stuck on the surface of the solid stationary phase.

A
A
Free

B
B

Stationary
Mobile Phase Phase
The equilibrium between the free and absorbed states depends on
three factors:
• the polarity and size of the molecule
• the polarity of the stationary phase
• the polarity of the solvent

Different molecules partition differently between the free and

absorbed state, that is the equilibria between these two states is not
the same.
• In TLC, the stationary phase is typically alumina (Al2O3.xH2O)n or
silica gel (SiO2.xH2O)n. The covalent network of these absorbents
create a very polar material.
• Silica gel is a granular, vitreous, porous form of silicon dioxide made
synthetically from sodium silicate. It is a naturally occurring mineral
that is purified and processed into either granular or beaded form. As
a desiccant, it has an average pore size of 2.4 nanometers and has a
strong affinity for water molecules.
The electropositive character of the aluminum or silicon and the
electronegative oxygen create a very polar stationary phase. Therefore,
more polar the molecule to be separated, the stronger is the attractive
force to the stationary phase.
The polar stationary phase will strongly attract like or polar molecules.
Nonpolar molecules will have a lower affinity for the stationary phase
and will remain longer in the solvent.
Procedure

Coat silica gel on the plate

Mark a line 2cm above the edge of the plate with pencil
Spot the sample and dry it
Place the slide in the beaker with solvent. Do not allow the spot to
dissolve in the solvent (mobile phase)
Cover the beaker with a lid
Take the slide out of the beaker before the solvent front reaches the
opposite edge.
Visualize the slide for result analysis and Rf value calculation.
 LANES

A B C SOLVENT FRONT

TLC PLATE

POINT OF ORIGIN

SOLVENT LEVEL
Visualization

• UV light
• Fluorescence
• Indicator dyes
Rf Values

TLC provide a chromatographic measurement known as

an Rf value. The Rf value is the “retardation factor” or
the “ratio-to-front” value expressed as a decimal
fraction.
Rf = distance spot travels/ distance solvent travels
Applications

• The qualitative testing of Various medicines such as sedatives, local anesthetics,

anticonvulsant tranquilizers, analgesics, antihistamines, steroids, hypnotics is done
by TLC.
• TLC is extremely useful in Biochemical analysis such as separation or isolation of
biochemical metabolites from its blood plasma, urine, body fluids, serum, etc.
• Thin layer chromatography can be used to identify natural products like essential
oils or volatile oil, fixed oil, glycosides, waxes, alkaloids, etc
• It is widely used in separating multicomponent pharmaceutical formulations.
• It is used to purify of any sample and direct comparison is done between the
sample and the authentic sample
• It is used in the food industry, to separate and identify colours, sweetening agent,
and preservatives
• It is used in the cosmetic industry.
• It is used to study if a reaction is complete.
Disadvantages:

• Thin Layer Chromatography plates do not have longer stationary phase.

• When compared to other chromatographic techniques the length of
separation is limited.
• The results generated from TLC are difficult to reproduce.
• Since TLC operates as an open system, some factors such as humidity and
temperature can be consequences to the outcome of the chromatogram.
• The detection limit is high and therefore if you want a lower detection
limit, you cannot use TLC.
• It is only a qualitative analysis technique and not quantitative.
Reference:

• https://www.youtube.com/watch?v=U2BKeT8toLQ
• https://www.youtube.com/watch?v=qdmKGskCyh8