Thin-layer

chromatography

Thin-layer chromatography (TLC) is a

chromatography technique that separates
components in non-volatile mixtures.[1]
 Thin-layer chromatography

Separation of black ink on a TLC plate

Acronym TLC

Classification Chromatography

Other techniques

Related HPTLC
Paper
chromatography
Agarose gel
electrophoresis
SDS-PAGE
It is performed on a TLC plate made up of
a non-reactive solid coated with a thin
layer of adsorbent material.[2] This is
called the stationary phase.[2] The sample
is deposited on the plate, which is eluted
with a solvent or solvent mixture known as
the mobile phase (or eluent).[3] This
solvent then moves up the plate via
capillary action.[4]As with all
chromatography, some compounds are
more attracted to the mobile phase, while
others are more attracted to the stationary
phase.[5] Therefore, different compounds
move up the TLC plate at different speeds
and become separated.[6] To visualize
colourless compounds, the plate is viewed
under UV-light or is stained.[7] Testing
different stationary and mobile phases is
often necessary to obtain well-defined and
separated spots.

TLC is quick, simple, and gives high

sensitivity for a relatively low cost.[5] It can
monitor reaction progress, identify
compounds in a mixture, determine purity,
or purify small amounts of compound.[5]

Procedure
The process for TLC is similar to paper
chromatography but provides faster runs,
better separations, and the choice
between different stationary phases.[5]
Plates can be labelled before or after the
chromatography process with a pencil or
other implement that will not interfere with
the process.[8]

There are four main stages to running a

thin-layer chromatography plate:[3][8]

Plate preparation: Using a capillary tube, a

small amount of a concentrated solution
of the sample is deposited near the
bottom edge of a TLC plate. Solvent
completely evaporate before the next step.
A vacuum chamber may be necessary for
non-volatile solvents. To make sure there
is sufficient compound to obtain a visible
result, the spotting procedure can be
repeated. Depending on the application,
they may also place different samples in a
row, the same distance from the bottom
edge; Each sample will move up the plate
in its own "lane."

TLC of three amino acids and a sample (left) with an English translation (right)

Development chamber preparation: The

solvent or solvent mixture is placed into a
transparent container
(separation/development chamber) to a
depth of less than 1 centimetre. They then
place a strip of filter paper (aka "wick")
along the container wall. This filter paper
should touch the solvent and almost reach
the top of the container. The container is
covered with a lid and the solvent vapors
are allowed to saturated the atmosphere
of the container. Failure to do so results in
poor separation and non-reproducible
results.

Development: The TLC plate is placed in

the container such that the sample spot(s)
are not submerged into the mobile phase.
The container is covered to prevent
solvent evaporation. The solvent migrates
up the plate by capillary action, meets the
sample mixture, and carries it up the plate
(elutes the sample). The plate is removed
from the container before the solvent
reaches the top of the plate. Otherwise,
the results will be misleading. Without
delay, they mark the solvent front: the
furthest extent of solvent up the plate.

Visualization: The solvent evaporate from

the plate. Visualization methods include
UV-light, staining, and many more.
Separation process and
principle
The separation of compounds is due to
the differences in their attraction to the
stationary phase and because of
differences in solubility in the solvent.[9] As
a result, the compounds and the mobile
phase compete for binding sites on the
stationary phase.[9] Different compounds
in the sample mixture travel at different
rates due to the differences in their
partition coefficients.[10] Different solvents,
or different solvent mixtures, gives
different separation.[5][11] The retardation
factor (Rf), or retention factor, quantifies
the results. It is the distance travelled by a
given substance divided by the distance
travelled by the mobile phase.

Development of a TLC plate. A spot that appears purple separates into a red and blue spot.

In normal-phase TLC, the stationary phase

is polar. Silica gel is very common in
normal-phase TLC. More polar compounds
in a sample mixture interact more strongly
with the polar stationary phase. As a
result, more polar compounds move less
(resulting in smaller Rf) while less polar
compounds move higher up the plate
(higher Rf).[10] A more polar mobile phase
also binds more strongly to the plate,
competing more with the compound for
binding sites; A more polar mobile phase
also dissolves polar compounds more.[10]
As such, all compounds on the TLC plate
move higher up the plate in polar solvent
mixtures. "Strong" solvents move
compounds higher up the plate, whereas
"weak" solvents move them less.[12]

If the stationary phase is non-polar, like

C18-functionalized silica plates, it is called
reverse-phase TLC. In this case, non-polar
compounds move less and polar
compounds move more. The solvent
mixture will also be much more polar than
in normal-phase TLC.[12]

Solvent choice

An eluotropic series, which orders solvents

by how much they move compounds, can
help in selecting a mobile phase.[5]
Solvents are also divided into solvent
selectivity groups.[11][13] Using solvents
with different elution strengths or different
selectivity groups can often give very
different results.[11][13] While single-solvent
mobile phases can sometimes give good
separation, some cases may require
solvent mixtures.[14]

In normal-phase TLC, the most common

solvent mixtures include ethyl
acetate/hexanes (EtOAc/Hex) for less
polar compounds and
methanol/dichloromethane (MeOH/DCM)
for more polar compounds.[15] Different
solvent mixtures and solvent ratios can
help give better separation.[16] In reverse-
phase TLC, solvent mixtures are typically
water with a less-polar solvent: Typical
choices are water with tetrahydrofuran
(THF), acetonitrile (ACN), or methanol.[15]
Analysis

TLC plate visualized with UV-light

As the chemicals being separated may be

colourless, several methods exist to
visualize the spots:

Placing the plate under blacklight

(366 nm light) makes fluorescent
compounds glow

TLC plates containing a small amount of

fluorescent compound (usually
manganese-activated zinc silicate) in
the adsorbent layer allow for
visualization of some compounds under
UV-C light (254 nm). The adsorbent layer
will fluoresce light-green, while spots
containing compounds that absorb UV-C
light will not.[4]

Placing the plate in a container filled

with Iodine vapours temporarily stains
the spots.[4] They typically become a
yellow or brown colour.
The TLC plate can either be dipped in or
sprayed with a stain. Many stains exist
but some examples include: [7][17][18]
Potassium permanganate

Bromine

Acidic vanillin

Phosphomolybdic acid
In the case of lipids, the chromatogram
may be transferred to a polyvinylidene
fluoride membrane and then subjected
to further analysis, for example, mass
spectrometry. This technique is known
as far-eastern blot.[4]
Plate production
TLC plates are usually commercially
available, with standard particle size
ranges to improve reproducibility.[19] They
are prepared by mixing the adsorbent,
such as silica gel, with a small amount of
inert binder like calcium sulfate (gypsum)
and water.[20] 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.[20] 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
TLC.[21]Other adsorbent coatings include
aluminium oxide (alumina), or cellulose.[20]

Applications

Reaction monitoring and

characterization

TLC is a useful tool for reaction

monitoring.[16] For this, the plate normally
contains a spot of starting material, a spot
from the reaction mixture, and a co-spot
(or cross-spot) containing both.[4][15] The
analysis will show if the starting material
disappeared and if any new products
appeared.[15] This provides a quick and
easy way to estimate how far a reaction
has proceeded. In one study, TLC has been
applied in the screening of organic
reactions. [22] The researchers react an
alcohol and a catalyst directly in the co-
spot of a TLC plate before developing it.
This provides quick and easy small-scale
testing of different reagents.

TLC for reaction monitoring and choosing a purification solvent mixture (left)TLC from the resulting flash column
chromatography (right)
Compound characterization with TLC is
also possible and is similar to reaction
monitoring. However, rather than spotting
with starting material and reaction
mixture, it is with an unknown and a
known compound. They may be the same
compound if both spots have the same Rf
and look the same under the chosen
visualization method. However, co-elution
complicates both reaction monitoring and
characterization. This is because different
compounds will move to the same spot on
the plate. In such cases, different solvent
mixtures may provide better separation.[23]
Purity and purification

TLC helps show the purity of a sample. A

pure sample should only contain one spot
by TLC. TLC is also useful for small-scale
purification.[24] Because the separated
compounds will be on different areas of
the plate, a scientist can scrape off the
stationary phase particles containing the
desired compound and dissolve them into
an appropriate solvent.[24] Once all the
compound dissolves in the solvent, they
filter out the silica particles, then
evaporate the solvent to isolate the
product. Big preparative TLC plates with
thick silica gel coatings can separate more
than 100 mg of material.[24]

For larger-scale purification and isolation,

TLC is useful to quickly test solvent
mixtures before running flash column
chromatography on a large batch of
impure material.[14][25] A compound elutes
from a column when the amount of
solvent collected is equal to 1/Rf.[26] The
eluent from flash column chromatography
gets collected across several containers
(for example, test tubes) called fractions.
TLC helps show which fractions contain
impurities and which contain pure
compound.
Furthermore, two-dimensional TLC[19] can
help check if a compound is stable on a
particular stationary phase. This test
requires two runs on a square-shaped TLC
plate. The plate is rotated by 90º before
the second run. If the target compound
appears on the diagonal of the square, it is
stable on the chosen stationary phase.
Otherwise, it is decomposing on the plate.
If this is the case, an alternative stationary
phase may prevent this decomposition.[27]

TLC is also an analytical method for the

direct separation of enantiomers and the
control of enantiomeric purity, e.g. active
pharmaceutical ingredients (APIs) that are
chiral.[28]

Separation of green plant matter in

spinach (note that images from steps 1-6
are zoomed into the bottom of the plate)
Step 1

Step 2
Step 3

Step 4
Step 5

Step 6
 Step 7

See also
Radial chromatography

HPTLC

Column chromatography

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Bibliography
F. Geiss (1987): Fundamentals of thin layer
chromatography planar chromatography,
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Justus G. Kirchner (1978): Thin-layer

chromatography, 2nd edition, Wiley

Joseph Sherma, Bernard Fried (1991):

Handbook of Thin-Layer Chromatography (=
Chromatographic Science. Bd. 55). Marcel
Dekker, New York NY, ISBN 0-8247-8335-2.
 Elke Hahn-Deinstorp: Applied Thin-Layer
Chromatography. Best Practice and
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u. a. 2000, ISBN 3-527-29839-8

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