Msc Medical Biochemistry,
Ph.D Research scholar.
Izmailov & Shraiber separated plant extracts
using 2mm thick and firm layer of alumina set
on glass plate.
In 1944, Consden, Gorden & Martin used filter
papers for separating amino acids.
In 1950, Kirchner identified terpenes on filter
In 1958, Stahl developed standard equipment
for analysing byTLC.
Separation based on the affinity of the compounds
towards the stationary phase.
Stationary phase: thin layer of adsorbent coated on
Mobile phase solvent flows through b’cos of
capillary action (against gravity)
The components with more affinity towards the
stationary phase travels slower.
Lesser affinity towards the stationary phase travels
1. Simple method & less expensive
2. Rapid technique
3. Separation upto µgms
4.Any type of compound can be analysed
5. Efficiency of separation: depends upon the particle size (
small) flow rate ( less viscous)
6. Detection is easy and not tedious
7. Capacity of the thin layer can be altered.
8. Needs less solvent, stationary phase & time
9. Corrosive spray reagents can be used without damaging
Preparation and activation ofTLC plates
Application of sample
Detecting or visualising agents
NAME COMPOSITION ADSORBENT:
Silicagel H Silicagel without
Silicagel G Silicagel +CaSO4 1:2
Silicagel GF Silicagel+Binder+f
Al2O3 G Al2O3 + binder 1:2
earth + binder
Glass plates 20cmX20cm(full plate), half plate
(20x10), quarter plate (20x5)
These dimensions are used since the width of the
TLC spreader is 20cm
Microscopic slides can be used for some
applications like monitoring the progress of a
The development time is 5 min
Good quality & should withstand temperatures used
for drying the plates
1. Pouring: slurry is prepared & poured on glass plate &
Demerit: uniformity in thickness cannot be ensured
2. Dipping: two plates are dipped into the slurry and are
separated after removing from slurry and later dried.
Demerit: larger quantity of slurry is needed
3. Spraying: using a sprayer
Demerit: layer thickness cannot be maintained uniformly
4. Spreading : the best technique
Normal thickness 0.25mm for analytical purposes
2mm thickness for preparative purposes
Oven temperature for drying should be 100-120 for 1 hr.
To get good spots, the conc of the sample has
to be minimum
2-5µl of a 1% solution is spotted using a
capillary tube or micropipette.
Spots should be kept atleast 2cm above the
base of the plate
Spotting area should not be immersed in the
Glass beakers, specimen jars
The tank should be lined inside with filter
paper moistened with mobile phase so as to
saturate the atmosphere
If saturation is not done, edge effect occurs
where the solvent front in the middle ofTLC
plate moves faster than that of the edge.
The solvent or mobile phase used depends upon
1. nature of the substances to be separated
2. nature of the stationary phase used
3. mode of chromatography ( normal / reverse
4. separation to be achieved ( analytical /
Eg: petroleum ether, CCl4, cyclohexane, CS2, Ether,
acetone, Benzene,Toluene, Ethylacetate, CHCl3,
alcohols, water, pyridine, organic acids,
1. One dimensional development (vertical)
2.Two dimensional development
3. Horizontal development
4. Multiple development
One dimensional development:The plates are kept vertical &
the solvent flows against gravity b’cos of capillary action.
Two dimensional development:
This tech is used for complex mixtures
First, the plates are developed in one axis & the plates after
drying are developed in the other axis.
After the development ofTLC plates, the spots
should be visualised.
Detecting coloured spots can be done visually.
For colourless spots
1. Non-specific methods: no. of spots can be
detected, but not the exact nature or type of
2. Specific methods: specific spray reagents or
detecting agents or visualising agents are used to
find out the nature of compounds or for
Where the no. of spots can be detected but not the
exact nature / type of compound
1. Iodine chamber method: where brown or amber
spots are observed
2. H2SO4 spray reagent: 70-80% of H2SO4 with few
mg of either K2Cr2O7 or KMnO4 or few ml of HNO3
as oxidising agent is used.
After spraying onTLC plates is heated in an oven
Black spots are seen due to charring of compounds
3. UV chamber for fluorescent compounds: when
compounds are viewed under UV chamber, at
254nm (short wavelength) or at 365nm (longer)
fluorescent compounds can be detected. Bright
spots are seen under a dark background.
4. Using fluorescent stationary phase: when the
compounds are non fluorescent, a fluorescent
stationary phase is used.When the plates are
viewed under UV chamber, dark spots are seen on a
fluorescent background. Eg: such stationary phase
is Silicagel GF
Specific spray reagents or detecting agents or
visualising agents are used to find out the nature of
compounds or for identification purposes.
Ex: 1. Ninhydrin for amino acids
2. FeCl3 phenolic compounds & tannins
3. Dragendroff’s reagent for alkaloids
4. 3,5- Dinitrobenzoic acid cardiac glycosides
5. 2,4-Dinitorphenylhydrazine aldehydes &
1. Destructive technique: samples are
Specific spray reagents, con H2SO4
2. Non-Destructive technique: Sample is not
destroyed even after detection.This is used in
Eg: UV-chamber method, Iodine chamber
method, densitometric method
Measures the density of the spots
When the optical density of the spots for the
standard & the test solution are measured ,
the quantity of the substance can be
The plates are neither destroyed nor eluted
with solvents to get the compounds.
This method is also called in-situ method
1. Separation of mixtures of drugs of chemical or
biological origin, plant extracts
2. Separation of carbohydrates, vitamins,
antibiotics, proteins, alkaloids, glycosides
3. identification of drugs
4. identification of related compounds in drugs
5. to detect the presence of foreign substances in
6. to detect decomposition products in drugs
HPTLC is a sophisticated & automated form ofTLC.
1. the use of precoated plates with stationary phase particle size of <10µ
2. wide choice of stationary phases like Silicagel, for normal phase
3. Auto sampler instead of manual spotting and streaking for preparative
4. New type of development chambers which requires less amt of solvents
5. More efficiency b’cos smaller & uniform size of adsorbents
6.The use of UV/Vis/Fluorescence scanner which scans the entire
chromatogram qualitatively & quantitatively.
The scanner is an advanced type of densitometer.
7. Increased data processing capabilities by the use of computers.
Rf value is the ratio of distance travelled by the
solute to the distance travelled by the solvent front.
The Rf value ranges from 0 to 1.0 (0.3-0.8)
Rx value: is nothing but the ratio of distance
travelled by the sample and the distance travelled
by the standard.
Rx value is always closer to 1.0
Rm value is used in qualitative analysis to find out
whether the compounds belong to a homologous