Thin Layer Chromatography
A little history
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To write with colors -- literally translated from its Greek
roots chroma and graphein , chromatography was first
developed by the Russian botanist Mikhail Tswett in 1903
as he produced a colorful separation of plant pigments
through a column of calcium carbonate, particularly the
carotenoids and the chlorophylls.
Early work consisted of dropping dyes onto filter paper
and seeing the development of concentric rings with
different colors: this technique was called “capillary
analysis”.
More history
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Enter Martin and Synge: these two British gentlemen
were pioneers in early separation science. They used silica
as a stationary phase with chloroform as the mobile
phase to separate amino acids in wool. They called their
idea “partition chromatography” and moved into the
theoretical by suggesting that the mobile phase could well
be a gas . . . Initial gas chromatography worked was
started during WW II.
For their efforts, they were awarded the Nobel Prize in
Chemistry and Physics in 1952.
More history
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Problems still were present: reproducibility and lack of
uniformity of silica production made the techniques less
than desirable.
Paper Chromatography was suggested as a way to
separate materials: cellulose with tightly adsorbed water
molecules would make a very uniform polar stationary
phase with less polar mobile phases. This technique
revolutionized planar chromatographic methods but had
limitations.
More history
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To overcome these limitations (stronger solvent systems
and developing agents), two Soviet pharmacists, Nikolay A.
Izmaylov and Maria S. Shrayber, coated films of silica onto
glass slides and used in a similar fashion to paper
chromatography.
In the 1950’s, a German chemist named Egan Stahl did a
HUGE amount of research into this field and produced
the “bible” of thin layer chromatography. Stahl’s book
emphasized applications and different reagents that could
be used to indicate types of compounds that were being
analyzed. He also helped standardize silica gel production,
which improved reproducibility!
So what’s going on in TLC?
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We have a backing – plastic, glass, aluminum
We have a stationary phase – usually silica, but
occasionally alumina, zirconium oxide or cellulose.
A binder holds the stationary phase to the backing (as an
example, gypsum is used to bind silica to the plate).
Within the stationary phase, there is usually a fluorescent
material added to allow easier visualization of separated
materials once the plate has been developed.
Theory
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If we thought about how a solute compound would interact
with such a solid stationary phase, we would realize that it
must essentially “stick” to the surface by some intermolecular
(van der Waal) forces. This sticking process in chromatography
is known as adsorption.
If a solute molecule dissolves into a liquid stationary phase in a
chromatographic system, we say that the molecule partitions
between the two phases.
These two terms are so important in chromatographic
systems that it is worth summarizing them again:
Adsorption – describes the process of a solute
molecule adhering to a solid surface
Partition – describes the process of a solute molecule
dissolving into a liquid stationary phase
Theory
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The van Deemter equation relates the resolving power (HETP,
height equivalent to a theoretical plate) of a chromatographic
column to the various flow and kinetic parameters which cause
peak broadening, as follows:
 HETP = A + B/u + C*u
Where HETP = height equivalent to a theoretical plate, a measure
of the resolving power of the column [m]
A = Eddy-diffusion parameter, related to channeling through a nonideal packing [m]
B = diffusion coefficient of the eluting particles in the longitudinal
direction, resulting in dispersion [m2 s-1]
C = Resistance to mass transfer coefficient of the analyte between
mobile and stationary phase [s]
u = Linear Velocity [m s-1]
In TLC, B predominates. u is really small (5-10 minutes to develop a
7.5 cm plate!)
Theory
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Smaller uniform particles provide lots of surfaces for the
two processes (adsorption and partition) to occur.
These processes combined with the capillary action of
the plate interacting with the solvent allows for “random
walk”.
The primary process at work for this kind of separation is
polarity: the stationary phase is usually polar and the
mobile phase is less polar (hexanes  methanol).
Theory to Practical
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As with most liquid chromatographic techniques, this
technique is SCALABLE! If you have a solvent system that
works for separating materials on an analytical level, using
a larger (preparative) plate or a column with a similar
silica (size of particle and pore size) will allow you to
scale from micrograms to 10-100’s of milligrams and in
some cases, gram separations.
Preparative TLC plates use larger tanks.
Prep TLC
Practical Considerations
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TLC plates need to be kept DRY. Remember that silica gel
is in those little packets you get with your new shoes or
electronics that says “do not eat!”.
Gentle heating (80 oC will remove any extra adsorbed
water).
TLC plates should only be handled on the edges since
your fingers contain oils which will interact/interfere with
the desired separation.
Proper application of the material for separation is
REALLY important.
How-to guide
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Prepare the solvent tank:
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Get your solvent system measured out.
Add a piece of filter paper to the back of the beaker/tank.
Add the solvent and tightly cover the tank and let the system
sit for a few minutes: this allows the tank to become saturated
with solvent vapors .
Prepare the TLC plate.
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Get a TLC plate and mark a line WITH PENCIL about 6-8 mm
above the depth of the solvent in the tank. This is the origin
Using a capillary tube, apply the materials to be separated on
the origin.
Dry off any solvent and GENTLY place the TLC plate into the
tank.
TLC chamber for
development e.g.
beaker
after ~5 min
with a lid or a
closed jar
after ~10 min
after drying
After the TLC plate has been run . . .
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Visualize the spots
Depending upon the materials present, you could try
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UV light (254 nm and/or 365 nm)
Reagent
Detection of
Aniline phthalate
reducing sugars, oxohalic acids
Bromocresol green
organic acids
2',7'-Dichlorofluorescein
4-(Dimethylamino)-benzaldehyde
Reagent according to
Dragendorff-Munier
lipids (saturated, unsaturated)
terpenes, sugars, steroids
alkaloids + other nitrogen compounds
Iron(III) chloride
Potassium hexacyanoferrate(III)
Molybdatophosphoric acid
acetylsalicylic acid, paracetamol
lipids, sterols, steroids,
reducing compounds
Ninhydrin
Rhodamin B
Rubeanic acid
amino acids, amines, amino sugars
lipids
heavy metal cations
http://www.mn-net.com/tabid/5578/default.aspx
Measuring the compounds
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The distance the solvent travels is measured from the
origin to the solvent front, which is marked as soon as
the TLC plate is removed from the TLC tank. This is Z.
The distance from the origin to the center of a spot is X.
Rf = X / Z, which is a unitless quantity.
Each compound should have a different Rf
Troubleshooting if your separation wasn’t
great
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Streaking of material: two usual causes. Either over
spotting/exceeding capacity of what plate can separate
OR adjusting the pH. Amines can streak less if a little
ammonium hydroxide is added to the solvent system.
Spots too faint: several possible causes. The material
spotted was too dilute, so concentrate and run another
TLC plate. Using the wrong reagent to visualize the spots
or this is the wrong solvent system for this plate.
Spots narrow as the plate develops: usually indicates that
the TLC plate was touching the filter paper/another plate
so capillary action wasn’t uniform.
So who uses TLC?
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Researchers – great for monitoring chemical reactions in
real time. As a reaction proceeds, the product appears on
the plate as a spot with a different Rf and the product
becomes more faint as time moves forward.
Forensic Analysts - TLC is useful in detecting chemicals of
forensic concern, including chemical weapons, explosives,
and illicit drugs. Unknown samples are compared to a
reference on the same TLC plate. If the unknown
compound has the same Rf and responds to a developing
reagent in the same manner as a known reference
compound like cocaine, a more careful analysis would be
warranted. The TLC could be run in 30 minutes!
So who uses TLC?
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Environmental Analysis
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Groundwater analysis, determination of pollutants from
abandoned armaments in soils and surface waters, decomposition
products from azo dyes used in textiles.
http://www.chem-ilp.net/labTechniques/TLC.htm
Other Planar Techniques
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Chromatotron