It is defined as the method of separation of a mixture into an
individual components through equilibrium distribution between
Michael tswett is being the father of liquid chromatography.
Tswett developed his idea in early 1900s.
4. • The chromatographic method of separation in general involve
in the following steps:-
Adsorption or retention of substance, or substance on
Separation of absorbed substance by mobile phase.
Recovery of separated substance by continues flow of the
mobile phase the method being called elution .
Qualitative and quantitative analysis of eluated substance.
5. THIN LAYER CHROMATOGRAPHY
It is a form of liquid chromatography consists of :-
A mobile phase (developing solvent).
A stationary phase ( a plate or strip coated with a form with a
silica gel ) .
Analysis is performed on a flat surface under atmospheric
pressure or room temperature.
6. The two most commonly types of thin layer chromatography
• Normal phase
• Reverse phase
• Normal phase :-
It is terminology used when the stationary phase is polar for
e.g. silica gel and the mobile phase is organic solvent of
mixture of organic solvents which is less polar than stationary
7. • In Reverse phase :-
In the reverse phase when the stationary phase is a silica
bonded with an organic substrate such as a long chain aliphatic
acid . Mobile phase is mixture of water and organic solvent
which is more polar than stationary phase.
• TLC technique involves the distribution of components of a
mixture to be separated between two phases.
• The components of the mixture are partitioned between an
adsorbent (stationary phase), and a solvent (mobile phase).
• Different compounds will have different solubility and
adsorption to the two phases between which they are to be
• In TLC separation of the individual substances is based on
their relative affinities towards stationary and mobile phases.
9. • The stationary phase: is a thin layer of adsorbent (usually
silica gel or alumina) coated on a plate.
• The mobile phase: is a developing liquid which flows through
the stationary phase, carrying the samples with it.
• Components with more affinity towards stationary phase
• Components with less affinity towards stationary phase
• Adsorbents used as Stationary Phase:
- Inorganic: Silica Gel, Aluminium Silicate, Bentonite.
-Organic: Cellulose & its acetylates, Charcoal & activated
Charcoal, Dextran Gel, Polyamides.
• Solvents used as Mobile Phase:
- Petroleum ether, Benzene, Carbon tetrachloride.
• Selection of Adsorbents and Solvents:
-Adsorbent should not adhere to glass plate.
-Solvents should be of high purity.
-Selected on the nature of compound to be separated(polar
or non polar).
11. TLC TECHNIQUE
Step1. Preparation of Slurry
• A plastic, glass or aluminum sheet is coated with a thin layer of
silica gel (adsorbent).
• Plates must be dried, activated and stored in desicator until used
Step 2. Preparation of tank
• Solvent mixtures should be freshly prepared for analysis.
• Solvent is poured down side of the tank (1.5cm depth).
• Tank is covered with the glass lid and kept for saturation.
12. Step 3. Application of sample (spot).
• A very small amount of sample (solution) to be analyzed is
applied in a small spot with a capillary tube, ~1cm from the
bottom of the TLC plate.
13. EXPERIMENTAL TECHNIQUE OF TLC
• The TLC is developed in a chamber which contains the mobile
• When the mobile phase rises up the plate up by capillary
action, the components dissolve in the solvent and move.
14. TLC TECHNIQUE
• Individual components in the
sample move up at different rates.
• More polar analytes interact more
strongly with the stationary phase
move very slowly up.
• More nonpolar analytes interact
less strongly with the polar silica
gel and more strongly with the less
polar mobile phase move higher
• Once the solvent reaches the top
(below ~1-2 cm) of the TLC sheet
the plate is removed from the
developing chamber and position
of solvent front is marked.
15. TLC TECHNIQUE
• The solvent is allowed to
evaporate from the TLC sheet.
• As the compound is colorless,
it can be visualized by suitable
Lipids - Iodine vapors
Amino acids - Ninhydrin
• Also, manganese-activated zinc
compound), is added to the
adsorbent that allows the
visualization of spots under a
black light (UV254 lamp).
• Once visible, the Rf value of
each spot can be determined.
16. RF VALUE
• Rf value indicates the position of migrated spots on chromatogram.
• In TLC the results are represented by Rf value which represents the
migration of solute relative to the solvent front.
• The Rf value is calculated as:-
Rf = distance travelled by the solute
distance travelled by the solvent
18. IMPORTANCE OF RF VALUE
• Qualitative results of TLC
– Rf values of any component lies between 0 to 1.
– no more than two decimal places .
• Rf values can be used to aid in the identification of a
substance by comparison to standards.
• Comparison should be made only between spots on the
same sheet, run at the same time.
• Test the purity of the sample: Thin layer chromatography
helps to detect the purity of the sample by
direct comparison with the standard or authentic sample. Any
impurity in the sample shows up as extra spots in
• Identify the components: Thin layer chromatography can
purify, isolate and identify the natural products like volatile oil
or essential oil, fixed oil, waxes, terpenes, alkaloids,
glycosides, steroids etc. in the test samples.
20. • Biochemical analysis: Biochemical metabolites from the
body fluids, blood plasma, serum, urine etc. can be isolated
using thin layer chromatography.
• In chemistry:
TLC is used to separate and identify closely related
compounds or cations and anions in inorganic chemistry.
• Pharmaceutical industries utilize TLC technique for
qualitative analysis or detect impurities in various
medicines like hypnotics, sedatives, anticonvulsants,
tranquillisers, anti-histaminics, analgesics, local
anaesthetics, steroids, etc. Yet another important application
of TLC is to separate multi-component pharmaceutical
formulations into its individual components.
21. • In food and cosmetic industry:
Any artificial colour, preservatives, sweetening agent, and
other impurities in food and cosmetic products can be detected
and isolated by TLC technique.
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