Thin Layer

Chromatography
(TLC)
Submitted to: Dr. Noreen Khalid
Submitted by: Malja Rehman
M.Phil Pharmaceutical Chemistry
1st Semester

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 Introduction
• Thin Layer Chromatography is a technique used to isolate non-
volatile mixtures.
• TLC is a sophisticated method of separating mixtures of two
or more compounds. The separation is accomplished by the
distribution of the mixture between two phases: one that is
stationary and one that is moving.
• 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 aluminum oxide, cellulose, or silica
gel.

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 Principle

• Like other chromatographic techniques, thin-layer chromatography

(TLC) depends on the separation principle.
• The separation relies on the relative affinity of compounds
towards both the phases.
• The compounds in the mobile phase move over the surface of the
stationary phase. The movement occurs in such a way that the
compounds which have a higher affinity to the stationary phase
move slowly while the other compounds travel fast.
• On completion of the separation process, the individual components
from the mixture appear as spots at respective levels on the plates.
Their character and nature are identified by suitable detection
techniques.
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Components
• Thin Layer Chromatography Plates – ready-made plates are used which are
chemically inert and stable. The stationary phase is applied on its surface in
the form of a thin layer. The stationary phase on the plate has a fine particle
size and also has a uniform thickness.
• Thin Layer Chromatography Chamber – Chamber is used to develop
plates. It is responsible to keep a steady environment inside which will help in
developing spots. Also, it prevents the solvent evaporation and keeps the
entire process dust-free.
• Thin Layer Chromatography Mobile phase – Mobile phase is the one that
moves and consists of a solvent mixture or a solvent. This phase should be
particulate-free. The higher the quality of purity the development of spots is
better.
• Thin Layer Chromatography Filter Paper – It has to be placed inside the
chamber. It is moistened in the mobile phase.

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Procedure
• The stationary phase that is applied to the plate is made to dry and stabilize.
• To apply sample spots, thin marks are made at the bottom of the plate with
the help of a pencil.
• Apply sample solutions to the marked spots.
• Pour the mobile phase into the TLC chamber and to maintain equal
humidity, place a moistened filter paper in the mobile phase.
• Place the plate in the TLC chamber and close it with a lid. It is kept in such a
way that the sample faces the mobile phase.
• Immerse the plate for development. Remember to keep the sample spots
well above the level of the mobile phase. Do not immerse it in the solvent.
• Wait till the development of spots. Once the spots are developed, take out the
plates and dry them. The sample spots can be observed under a UV light
chamber.

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Spot measurement
Using fluorescence
• The stationary phase on a thin layer plate often has a substance added to it which
will fluoresce when exposed to UV light. That means that if you shine UV light on
it, it will glow.
• That glow is masked at the position where the spots are on the final chromatogram
- even if those spots are invisible to the eye. That means that if you shine UV light
on the plate, it will all glow apart from where the spots are. The spots show up as
darker patches.

While the UV is still shining on the plate,

you obviously have to mark the positions
of the spots by drawing a pencil circle
around them. As soon as you switch off
the UV source, the spots will disappear
again.

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Showing the spots up chemically
• In some cases, it may be possible to make the spots visible by
reacting them with something which produces a colored product. A
good example of this is in chromatograms produced from amino
acid mixtures.
• The chromatogram is allowed to dry and is then sprayed with a
solution of ninhydrin. Ninhydrin reacts with amino acids to give
colored compounds, mainly brown or purple.

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• In another method, the chromatogram is allowed to dry and
then placed in an enclosed container along with a few
iodine crystals.
• The iodine vapour in the container may either react with the
spots on the chromatogram, or simply stick more to the
spots than to the rest of the plate. Either way, the substances
you are interested in may show up as brownish spots.

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Measuring Rf values
• To know that how many different compounds are in mixtures, you have to
measure the distance travelled by the solvent, and the distance travelled by
individual spots.
• When the solvent front gets close to the top of the plate, the plate is removed from
the beaker and the position of the solvent is marked with another line before it has
a chance to evaporate.
• These measurements are then taken:
• The Rf value for each dye is then worked out using the formula:

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Common Problems
There are common problems in TLC that should be avoided.
Normally, these problems can be solved or avoided if taught proper
techniques.
• Over-large Spots: Spotting sizes of your sample should be not be
larger than 1-2 mm in diameter. The component spots will never be
larger than or smaller than your sample origin spot. If you have an
over-large spot, this could cause overlapping of other component
spots with similar Rf values on your TLC plate. If overlapping
occurs, it would prove difficult to resolve the different components.
• Uneven Advance of Solvent Front: Uneven advance of the mobile
phase is a common problem encountered in TLC. Consequences
would be inaccurate Rf values due to the uneven advance of sample
origin spots. This uneven advance can be caused by a few factors
listed below.
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1- No flat bottom. When placing the TLC plate into the
chamber, place the bottom of the plate on the edge of the
chamber (normally glass container (e.g. beaker)) and lean the
top of the plate along the other side of the chamber. Also,
make sure that the TLC plate is placed in the chamber evenly.
Do not tilt the plate or sit it at an angle.
2- Not enough solvent. There should be enough solvent
(depends on size of the chamber) to travel up the length of the
TLC plate.
3- Plate is not cut evenly. It is recommended that a ruler is
used so that the plate is cut evenly.
4- Rarely, water is used as a solvent because it produces an
uneven curve front which is mainly accounted for by its
surface tension.

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• Streaking: If the sample spot is too concentrated, the substance
will travel up the stationary phase as a streak rather than a single
separated spot.
In other words, the solvent can not handle the concentrated sample
and in result, moves as much of the substance as it can up the
stationary phase. The substance that it can not move is left behind.
This can be eliminated by diluting the sample solution. To ensure
that you have enough solution, use a short-wave UV light to see if
the spot is visible (normally purple in color), as stated earlier.
• Spotting: The sample should be above the solvent level. If the
solvent level covers the sample, the sample spot will be washed
off into the solvent before it travels up the TLC plate.

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Applications

• The qualitative testing of Various medicines such as sedatives, local

anaesthetics, anticonvulsant tranquilisers, analgesics, antihistamines,
steroids, hypnotics is done by TLC.
• To monitor the progress of a reaction, identify compounds present in
a given mixture, and determine the purity of a substance.
• 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.

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• It is widely used in separating multicomponent
pharmaceutical formulations.
• It is used to purify of any sample and direct comparison is
done between the sample and the authentic sample.
• It is used in the food industry, to separate and identify colors,
sweetening agent, and preservatives
• It is used in the cosmetic industry.
• It is used to study if a reaction is complete.

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Disadvantages
• Thin Layer Chromatography plates do not have longer stationary
phase.
• When compared to other chromatographic techniques the length of
separation is limited.
• The results generated from TLC are difficult to reproduce.
• Since TLC operates as an open system, some factors such as
humidity and temperature can be consequences to the final outcome
of the chromatogram.
• The detection limit is high and therefore if you want a lower
detection limit, you cannot use TLC.
• It is only a qualitative analysis technique and not quantitative.

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References
AN OVERVIEW ON THIN LAYER CHROMATOGRAPHY
http://dx.doi.org/10.13040/IJPSR.0975-8232.2(2).256-67
Thin-layer chromatography in medicinal chemistry
https://doi.org/10.1080/10826076.2019.1585615

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