THIN LAYER CHROMATOGRAPHY

Chromatography is the separation of a mixture into its individual components. All types of
chromatography works in the same principle. They all have a stationary phase (a solid or a
liquid supported on a solid most commonly Silica Gel TLC plates) and a mobile phase (a
liquid or a gas). The mobile phase flows through the stationary phase and carries the
components of the mixture with it. Different components travel at different rates. Thin layer
chromatography consists of a thin, uniform layer of silica gel or alumina coated onto a piece
of glass, metal or rigid plastic. The silica gel (or the alumina) is the stationary phase. The
stationary phase for thin layer chromatography also often contains a substance which
fluoresces in UV light. The mobile phase is a suitable liquid solvent or mixture of solvents.
5.5.1 Stationary phase
Silica gel is a form of silicon dioxide (silica). The surface of the
silica gel has Si-O-H bonds instead of Si-O-Si bonds. This make the
surface very polar and, because of the OH (hydroxyl) groups, and
form hydrogen bonds with suitable compounds around it as well as
Fig 5.5.1: Silica structure
van der Waals dispersion forces and dipole-dipole attractions.
5.5.2 Mobile Phase
Analyte

exists

in

equilibrium

between the stationary and mobile

phases. If there exists a greater
polar

attraction

between

the

Table 5.5.2 Solvents used for TLC

Nonpolar mobile
Chloroform, Methanol
phase(basic)
(19:1)
Polar basic
Ethyl acetate, Methanol,
Water (8:1.2:0.8)
solvent(neutral)

solvent and the analyte than the silica and the analyte, then the analyte will spend more time
traveling in the mobile phase along the. Thus, the greater the polarity of a solvent, greater
would be the elution. However, this is all relative to the polarity of the analyte itself. If the
analyte has a greater polarity than the solvent, it will remain more easily attached to the silica.
Two solvent systems were used with increasing polarity for proper separation of compounds
present in the aqueous and ethanol extracts.

5.5.3 Visualization
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Table 5.5.3 TLC Spotting Dye: Iodine Vapour

Compounds visualized
Unsaturated & Aromatic Compou
Preparation

Iodine crystals in an amber bottle

Visualization colour

Dark brown in light brown backgrou

*Iodine stain can be removed by heating.

TLC plates were removed from the TLC chamber when the solvent front reached 1cm below
the top of TLC plate. The plates were air dried at room temperature, viewed in visible light,
254nm ultraviolet, followed by staining in iodine vapours, and, finally viewed the iodine
stained chromatogram in 254nm ultraviolet.

5.5.4 Calculation of Retention factor

Retention factor (Rf)is defined as the distance travelled by the compound divided by the
distance travelled by the solvent front. The larger the Rf value of a compound, the less is the
polarity of the compound.
Distance travelled by the Analyte
Retention factor (Rf) =
Distance travelled by the solvent front

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Spotting of Extract on the TLC plate

1.
2.
3.
4.

Cut the TLC plates into desired sizes

Place the TLC plates on a evenly plane surface (table/bench top)
Take a clean and dry ruler (scale)
Draw a horizontal line touching one end to another with a pencil above 1cm from the

bottom of the TLC plates

5. Take clean and dry capillary tubes; dip its end into the extract
6. Place the tip of the loaded capillary tube on the TLC plate such that a tiny drop of the
extract is absorbed on the TLC plate
7. Let the drop air-dry
8. Repeat the above 2 steps for 3 times
Running the TLC system
1. Take a clean and dry TLC chamber (A large glass beaker can be used as a substitute)
2. Add solvents (Chloroform + Methanol 19:1 OR Ethyl acetate + Methanol + D/W
3.
4.
5.
6.

8:1.2:0.8)
Make sure the solvents cover about 0.5 cm of the TLC chamber
Put the TLC plates into the TLC chamber
Cover the TLC Chamber with aluminium foil (air proof)
Observe the TLC plate every 5 mins . Remove the TLC plate from the TLC chamber
once the solvent front reaches about 2 cms below the top

Visualization of the Plate

1.
2.
3.
4.
5.
6.

Take a clean and dry TLC chamber (A large glass beaker can be used as a substitute)
Add about a teaspoon full of Iodine crystals into the TLC chamber
Spread the Iodine crystals evenly in the bottom of the TLC chamber
Put the TLC plates into the TLC chmaber
Cover the TLC Chamber with aluminium foil (air proof)
Wait for 5 15 mins until spots develops on the TLC plates

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