Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The experiment is conducted on a sheet of aluminium foil, plastic, or glass which is coated with a thin layer of adsorbent material. The material usually used is aluminium oxide, cellulose, or silica gel.

2. The development chamber is a container
(jar, beaker, or flask) that can be sealed
tight enough that the atmosphere in the
container is saturated with the developing
liquid. Beakers and flasks can be sealed by
covering the opening with a watch glass,
parafilm, or aluminum foil.
To the developing chamber, add enough of
the appropriate developing liquid so that it
is 0.5 to 1 cm deep in the bottom. Next,
place a piece of filter paper into the
developing chamber so that it lines the
walls and is immersed in the liquid.
Close the developing chamber tightly, and
let it stand for a few minutes to allow the
atmosphere within the chamber to become
saturated with solvent vapor.

3. TLC plates are usually commercially available,
with standard particle size ranges to improve
reproducibility. They are prepared by mixing
the adsorbent, such as silica gel, with a small
amount of inert binder like calcium sulfate
(gypsum) and water. This mixture is spread as
a thick slurry on an unreactive carrier sheet,
usually glass, thick aluminum foil, or plastic.
The resultant plate is dried and activated by
heating in an oven for thirty minutes at 110 °C.
The thickness of the absorbent layer is
typically around 0.1–0.25 mm for analytical
purposes and around 0.5–2.0 mm for
preparative

4. .
Using TLC pipettes, apply spots
of analyte to the line. Make sure
enough sample is spotted on
the plate.
If the spot is not visible, more sample needs to be
applied to the plate

5. A TLC plate can be developed in a beaker or closed jar (see picture below).
Place a small amount of solvent (= mobile phase) in the container. The solvent
level has to be below the starting line of the TLC, otherwise the spots will
dissolve away.

6. The lower edge of the plate is then dipped in a solvent.
The solvent (eluent) travels up the matrix by capillarity, moving the
components of the samples at various rates because of their different
degrees of interaction with the matrix and solubility in the developing
solvent.
Non-polar solvents will force non-polar compounds to the top of the plate,
because the compounds dissolve well and do not interact with the polar
stationary phase.
Allow the solvent to travel up the plate until ~1 cm from the top.
Take the plate out and mark the solvent front immediately.
Do not allow the solvent to run over the edge of the plate. Next, let the
solvent evaporate completely.

7. If there are any colored
spots, circle them lightly
with a apencil.
most samples are not
co;ored and need to be
visualized with a UV
lamp.

8. Make sure you are
wearing goggles and
do not look directly
into the lamp. Protect
your skin by wearing
gloves.
Hold the UV lamp over the plate and circle any spots you see

9. Iodine
The plate can be stained with iodine.
This can be achieved rapidly, by shaking the plate in a bottle containing silica and a
few crystals of iodine.
The iodine will stain any compound that reacts with it and so is especially good for
visualizing unsaturated compounds.
Most spots show up within a few seconds, but the stain is not usually permanent.
UV light
The plate can be viewed under a ultraviolet lamp to show any uv-active spots.

10. Dipping Solutions:
The plate can be treated with one of the reagents listed below and then heated to stain the
spots. The reagent can be sprayed onto the plates, but this technique is quite hazardous and
it is more effective for them to be dipped in the reagent. To do this, first let the tlc solvent
evaporate, then holding the edge of the plate with tweezers, immerse the plate as
completely as possible in the stain and remove it quickly. Rest the edge of the plate on a
paper towel to absorb the excess stain before heating carefully on a hot plate or with a heat
gun, until the spots show. This method is always permanent and should be done last. When
glass plates are used the spots can sometimes be seen more clearly from the glass side of
the plate.

11. The qualitative testing of Various medicines such as sedatives, local
anaesthetics, anticonvulsant tranquilisers, 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 used to study if a reaction is complete.

12. multicomponent pharmaceutical
It is widely used in separating
formulations.
It is used for the purification of samples 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.