THIN LAYER

CHROMATOGRAPHY
Submitted by
Abhijit padhi
Chromatography is an important biophysical
technique that enables the separation, identification, and
purification of the components of a mixture for qualitative
and quantitative analysis.
In this physical method of separation, the components to be
separated are distributed between two phases, one of which
is stationary (stationary phase) while the other (the mobile
phase) moves in a definite direction. Depending upon the
stationary phase and mobile phase chosen, they can be of
different types.
What is Thin Layer
Chromatography (TLC)?
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
plate and liquid as a mobile phase.
Michael
tswett is credited
as being the father
of liquid
chromatography.
Tswett developed
his ideas in early
1900’s.
The two most common classes of TLC
are:-
-Normal phase
-Reversed phase
Normal phase
Normal phase is 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 a terminology used when the
stationary phase is a silica bounded 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.
Principle of Thin Layer Chromatography
(TLC)
•Thin-layer chromatography is performed on a sheet of glass,
plastic, or aluminum foil, which is coated with a thin layer
of adsorbent material, usually silica gel, aluminum oxide (alumina),
or cellulose. This layer of adsorbent is known as the stationary
phase.
•After the sample has been applied on the plate, a solvent or solvent
mixture (known as the mobile phase) is drawn up the plate
via capillary action.
•Once separation occurs, the individual components are visualized
as spots at a respective level of travel on the plate. Their nature or
character is identified by means of suitable detection techniques.
Components of Thin Layer Chromatography
(TLC)
TLC system components consists of:
1.TLC plates, preferably ready made with a stationary phase: These are stable
and chemically inert plates, where a thin layer of stationary phase is applied on
its whole surface layer. The stationary phase on the plates is of uniform thickness
and is in a fine particle size.
2.TLC chamber- This is used for the development of TLC plate. The chamber
maintains a uniform environment inside for proper development of spots. It also
prevents the evaporation of solvents, and keeps the process dust free.
3.Mobile phase- This comprises of a solvent or solvent mixture The mobile phase
used should be particulate-free and of the highest purity for proper development
of TLC spots. The solvents recommended are chemically inert with the sample, a
stationary phase.
4.A filter paper- This is moistened in the mobile phase, to be placed inside the
chamber. This helps develop a uniform rise in a mobile phase over the length of
the stationary phase.
Procedure of Thin Layer
Chromatography (TLC)
1.With a pencil, a thin mark is made at the bottom of the plate to apply the sample
spots.
2.Then, samples solutions are applied on the spots marked on the line in equal
distances.
3.The mobile phase is poured into the TLC chamber to a leveled few centimeters
above the chamber bottom.
4.A moistened filter paper in mobile phase is placed on the inner wall of the
chamber to maintain equal humidity (and also thereby avoids edge effect).
5.Now, the plate prepared with sample spotting is placed in TLC chamber so that
the side of the plate with the sample line is facing the mobile phase. Then the
chamber is closed with a lid.
6.The plate is then immersed, such that the sample spots are well above the level of
mobile phase (but not immersed in the solvent) for development.
7.Sufficient time is given for the development of spots.
8.The plates are then removed and allowed to dry.
9.The sample spots are then seen in a suitable UV light chamber, or any other
methods as recommended for the given sample.
PRACTICAL
REQUIREMENTS

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

2. GLASS PLATE
Specific dimensions-

20cm X 20cm, 20cm X 10cm, 20cm X 5cm

-Microscopic slides can also be used

-Plates should be of good quality & withstand high temperatures.

PREPARATION & ACTIVATION OF TLC PLATES

1.Pouring (simplest methods)

2.Dipping (used for small plates)

3.Spraying (difficult to get uniform layers)

4.Spreading (best technique) TLC Spreader

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....
THIN LAYER CHROMATOGRAPHY
DRYING OF CHROMATOGRAM

After the solvent has moved a certain distance for certain

time the chromatogram is taken out from the tank &
position of the solvent front is marked with a pencil.

They are dried by cold or hot air depending on volatility of

solvents. A simple hair dryer is a convenient device to dry
chromatograms.
THIN LAYER CHROMATOGRAPHY
DETECTING/VISUALISING AGENTS

If the substance are colored they are visually detected easily.

But for colorless substance, Physical and chemical methods are

used to detect the spot.

(a) Non specific methods (Physical methods)

E.g. iodine chamber method,

UV chamber for fluorescent compounds - at 254 or at 365nm.

(b) Specific methods (Chemical methods) or Spraying
method

EXAMPLES

Ferric chloride-Phenolic comp. & tannins

Ninhydrin in acetone-Amino acids

Dragendroff's reagent-Alkaloids

3,5 dinitro benzoic acid-Cardiac glycosides

QUANTITATIVE
ESTIMATIONS

The method can be divided into two main groups

1 Direct techniques-

2 Indirect techniques-
THIN LAYER CHROMATOGRAPHY
Direct Measurement Method

(i) Comparison of visible spots

A rough quantitative measurements Component in a mixture can

be carried out by comparing the intensity and size of the spot with
a standard substance.

(ii) Photo densitometry

The method is used with the chromatograms of colored compound,

instrument which measures quantitatively the density of the spots.
Retention Factor (Rf ) Value
•The behavior of a compound on a TLC is usually described in
terms of its relative mobility or Rf value.
•Rf or Retention factor is a unique value for each compound under
the same conditions.
•The Rf for a compound is a constant from one experiment to the
next only if the chromatography conditions below are also constant:
•solvent system
•adsorbent
•thickness of the adsorbent
•amount of material spotted
•temperature
•Since these factors are difficult to keep constant from experiment
to experiment, relative Rf values are generally considered.
•Relative Rf” means that the values are reported relative to a
standard.
Applications of Thin Layer
Chromatography (TLC)
1.In monitoring the progress of reactions
2.Identify compounds present in a given mixture
3.Determine the purity of a substance.
•Analyzing ceramides and fatty acids
•Detection of pesticides or insecticides in food and water
•Analyzing the dye composition of fibers in forensics
•Assaying the radiochemical purity of radiopharmaceuticals
•Identification of medicinal plants and their constituents.
Thank you