THIN LAYER CHROMATOGRAPHY

This page is an introduction to chromatography using thin layer

chromatography as an example. Although if you are a beginner
you may be more familiar with paper chromatography, thin layer
chromatography is equally easy to describe and more
straightforward to explain.


Note: I'm taking a simple view of the way that thin layer
chromatography works in terms of adsorption (see below)
which should be adequate for students doing courses for 16
- 18 year olds. The reality is more complicated and the
explanation will vary depending on what sort of solvent or
solvent mixture you are using. Some similar problems are
discussed on the page about paper chromatography, but I
am unwilling to do the same thing on this page which is
intended as a fairly gentle introduction to chromatography.


Carrying out thin layer chromatography
Background
Chromatography is used to separate mixtures of substances into
their components. All forms of chromatography work on the
same principle.
They all have a stationary phase (a solid, or a liquid supported
on a solid) and a mobile phase (a liquid or a gas). The mobile
phase flows through the stationary phase and carries the
components of the mixture with it. Different components travel at
different rates. We'll look at the reasons for this further down the
page.
Thin layer chromatography is done exactly as it says - using a
thin, uniform layer of silica gel or alumina coated onto a piece of
glass, metal or rigid plastic.
The silica gel (or the alumina) is the stationary phase. The
stationary phase for thin layer chromatography also often
contains a substance which fluoresces in UV light - for reasons
you will see later. The mobile phase is a suitable liquid solvent
or mixture of solvents.

Producing the chromatogram
We'll start with a very simple case - just trying to show that a
particular dye is in fact a mixture of simpler dyes.




Note: The chromatography plate will in fact be pure white -
not pale grey. I'm forced to show it as off-white because of
the way I construct the diagrams. Anything I draw as pure
white allows the background colour of the page to show
through.


A pencil line is drawn near the bottom of the plate and a small
drop of a solution of the dye mixture is placed on it. Any labelling
on the plate to show the original position of the drop must also
be in pencil. If any of this was done in ink, dyes from the ink
would also move as the chromatogram developed.
When the spot of mixture is dry, the plate is stood in a shallow
layer of solvent in a covered beaker. It is important that the
solvent level is below the line with the spot on it.
The reason for covering the beaker is to make sure that the
atmosphere in the beaker is saturated with solvent vapour. To
help this, the beaker is often lined with some filter paper soaked
in solvent. Saturating the atmosphere in the beaker with vapour
stops the solvent from evaporating as it rises up the plate.
As the solvent slowly travels up the plate, the different
components of the dye mixture travel at different rates and the
mixture is separated into different coloured spots.



The diagram shows the plate after the solvent has moved about
half way up it.
The solvent is allowed to rise until it almost reaches the top of
the plate. That will give the maximum separation of the dye
components for this particular combination of solvent and
stationary phase.

Measuring R
f
values
If all you wanted to know is how many different dyes made up
the mixture, you could just stop there. However, measurements
are often taken from the plate in order to help identify the
compounds present. These measurements are 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 R
f
value for each dye is then worked out using the formula:



For example, if the red component travelled 1.7 cm from the
base line while the solvent had travelled 5.0 cm, then the R
f

value for the red dye is:


If you could repeat this experiment under exactly the same
conditions, then the R
f
values for each dye would always be the
same. For example, the R
f
value for the red dye would always
be 0.34. However, if anything changes (the temperature, the
exact composition of the solvent, and so on), that is no longer
true. You have to bear this in mind if you want to use this
technique to identify a particular dye. We'll look at how you can
use thin layer chromatography for analysis further down the
page.

What if the substances you are interested in are colourless?
There are two simple ways of getting around this problem.
Using fluorescence
You may remember that I mentioned that 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.

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 coloured
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
coloured compounds, mainly brown or purple.



In another method, the chromatogram is again allowed to dry
and then placed in an enclosed container (such as another
beaker covered with a watch glass) 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.

Using thin layer chromatography to identify compounds
Suppose you had a mixture of amino acids and wanted to find
out which particular amino acids the mixture contained. For
simplicity we'll assume that you know the mixture can only
possibly contain five of the common amino acids.
A small drop of the mixture is placed on the base line of the thin
layer plate, and similar small spots of the known amino acids are
placed alongside it. The plate is then stood in a suitable solvent
and left to develop as before. In the diagram, the mixture is M,
and the known amino acids are labelled 1 to 5.
The left-hand diagram shows the plate after the solvent front has
almost reached the top. The spots are still invisible. The second
diagram shows what it might look like after spraying with
ninhydrin.



There is no need to measure the R
f
values because you can
easily compare the spots in the mixture with those of the known
amino acids - both from their positions and their colours.
In this example, the mixture contains the amino acids labelled as
1, 4 and 5.
And what if the mixture contained amino acids other than the
ones we have used for comparison? There would be spots in
the mixture which didn't match those from the known amino
acids. You would have to re-run the experiment using other
amino acids for comparison.

How does thin layer chromatography work?
The stationary phase - silica gel
Silica gel is a form of silicon dioxide (silica). The silicon atoms
are joined via oxygen atoms in a giant covalent structure.
However, at the surface of the silica gel, the silicon atoms are
attached to -OH groups.


Note: If you aren't sure about it, you will find one possible
structure of silicon dioxide towards the bottom of the page
you will get to by following this link.
Use the BACK button on your browser to return quickly to
this page.


So, at the surface of the silica gel you have Si-O-H bonds
instead of Si-O-Si bonds. The diagram shows a small part of the
silica surface.



The surface of the silica gel is very polar and, because of the -
OH groups, can form hydrogen bonds with suitable compounds
around it as well as van der Waals dispersion forces and dipole-
dipole attractions.


Note: If you aren't sure about hydrogen bonds and van der
Waals forces follow this link to the page about hydrogen
bonding. You will find a further link to van der Waals forces
at the bottom of that page.
Use the BACK button on your browser to return quickly to
this page.


The other commonly used stationary phase is alumina -
aluminium oxide. The aluminium atoms on the surface of this
also have -OH groups attached. Anything we say about silica gel
therefore applies equally to alumina.

What separates the compounds as a chromatogram
develops?
As the solvent begins to soak up the plate, it first dissolves the
compounds in the spot that you have put on the base line. The
compounds present will then tend to get carried up the
chromatography plate as the solvent continues to move
upwards.
How fast the compounds get carried up the plate depends on
two things:
How soluble the compound is in the solvent. This will
depend on how much attraction there is between the
molecules of the compound and those of the solvent.
How much the compound sticks to the stationary phase -
the silica gel, for example. This will depend on how much
attraction there is between the molecules of the
compound and the silica gel.
Suppose the original spot contained two compounds - one of
which can form hydrogen bonds, and one of which can only take
part in weaker van der Waals interactions.
The one which can hydrogen bond will stick to the surface of the
silica gel more firmly than the other one. We say that one is
adsorbed more strongly than the other. Adsorption is the name
given to one substance forming some sort of bonds to the
surface of another one.
Adsorption isn't permanent - there is a constant movement of a
molecule between being adsorbed onto the silica gel surface
and going back into solution in the solvent.
Obviously the compound can only travel up the plate during the
time that it is dissolved in the solvent. While it is adsorbed on the
silica gel, it is temporarily stopped - the solvent is moving on
without it. That means that the more strongly a compound is
adsorbed, the less distance it can travel up the plate.
In the example we started with, the compound which can
hydrogen bond will adsorb more strongly than the one
dependent on van der Waals interactions, and so won't travel so
far up the plate.
What if both components of the mixture can hydrogen bond?
It is very unlikely that both will hydrogen bond to exactly the
same extent, and be soluble in the solvent to exactly the same
extent. It isn't just the attraction of the compound for the silica
gel which matters. Attractions between the compound and the
solvent are also important - they will affect how easily the
compound is pulled back into solution away from the surface of
the silica.
However, it may be that the compounds don't separate out very
well when you make the chromatogram. In that case, changing
the solvent may well help - including perhaps changing the pH of
the solvent.
This is to some extent just a matter of trial and error - if one
solvent or solvent mixture doesn't work very well, you try another
one. (Or, more likely, given the level you are probably working
at, someone else has already done all the hard work for you,
and you just use the solvent mixture you are given and
everything will work perfectly!)












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About Us
Thin Layer Chromatography (TLC)
TLC is a simple, quick, and inexpensive procedure that gives the chemist a
quick answer as to how many components are in a mixture. TLC is also used
to support the identity of a compound in a mixture when the R
f
of a
compound is compared with the R
f
of a known compound (preferably both
run on the same TLC plate).
A TLC plate is a sheet of glass, metal, or plastic which is coated with a thin
layer of a solid adsorbent (usually silica or alumina). A small amount of the
mixture to be analyzed is spotted near the bottom of this plate. The TLC
plate is then placed in a shallow pool of a solvent in a developing chamber
so that only the very bottom of the plate is in the liquid. This liquid, or the
eluent, is the mobile phase, and it slowly rises up the TLC plate by capillary
action.
As the solvent moves past the spot that was applied, an equilibrium is
established for each component of the mixture between the molecules of
that component which are adsorbed on the solid and the molecules which
are in solution. In principle, the components will differ in solubility and in the
strength of their adsorption to the adsorbent and some components will be
carried farther up the plate than others. When the solvent has reached the
top of the plate, the plate is removed from the developing chamber, dried,
and the separated components of the mixture are visualized. If the
compounds are colored, visualization is straightforward. Usually the
compounds are not colored, so a UV lamp is used to visualize the plates.
(The plate itself contains a fluorescent dye which glows everywhere except
where an organic compound is on the plate.)
How To Run a TLC Plate

Step 1: Prepare the
developing container
The developing container
for TLC can be a specially
designed chamber, a jar
with a lid, or a beaker
with a watch glass on
the top (the latter is
used in the undergrad
labs at CU). Pour solvent
into the chamber to a
depth of just less than
0.5 cm. To aid in the
saturation of the TLC
chamber with solvent
vapors, you can line part
of the inside of the
beaker with filter paper.
Cover the beaker with a
watch glass, swirl it
gently, and allow it to
stand while you prepare
your TLC plate.

Step 2: Prepare the TLC plate
TLC plates used in the
organic chem teaching
labs are purchased as 5
cm x 20 cm sheets. Each
large sheet is cut
horizontally into plates
which are 5 cm tall by
various widths; the more
samples you plan to run
on a plate, the wider it
needs to be. Shown in
the photo to the left is a
box of TLC plates, a large
un-cut TLC sheet, and a
small TLC plate which
has been cut to a
convenient size. Handle
the plates carefully so
that you do not disturb
the coating of adsorbent
or get them dirty.

Measure 0.5 cm from the
bottom of the plate.
Using a pencil, draw a
line across the plate at
the 0.5 cm mark. This is
the origin: the line on
which you will spot the
plate. Take care not to
press so hard with the
pencil that you disturb
the adsorbent. Under the
line, mark lightly the
name of the samples you
will spot on the plate, or
mark numbers for time
points. Leave enough
space between the
samples so that they do
not run together; about 4
samples on a 5 cm wide
plate is advised.

Step 3: Spot the TLC plate
If the sample is not
already in solution,
dissolve about 1 mg in 1
mL of a volatile solvent
such as hexanes, ethyl
acetate, or methylene
chloride. As a rule of
thumb, a concentration
of 1% usually works well
for TLC analysis. If the
sample is too
concentrated, it will run
as a smear or streak (see
troubleshooting section
below); if it is not
concentrated enough,
you will see nothing on
the plate. Sometimes you
will need to use trial and
error to get well-sized,
easy to read spots.

Obtain a a
microcapillary. In the
organic teaching labs, we
use 10L microcaps -
they are easier to handle
than the smaller ones
used in research labs.
Dip the microcap into the
solution and then gently
touch the end of it onto
the proper location on
the TLC plate. Don't
allow the spot to become
too large - if necessary,
you can touch it to the
plate, lift it off and blow
on the spot. If you
repeat these steps, the
wet area on the plate will
stay small.

This example plate has
been spotted with three
different quantities of
the same solution and is
ready to develop. If you
are unsure of how much
sample to spot, you can
always spot multiple
quantities and see which
looks best.

Step 4: Develop the plate
Place the prepared TLC
plate in the developing
beaker, cover the beaker
with the watch glass,
and leave it undisturbed
on your bench top. The
solvent will rise up the
TLC plate by capillary
action. Make sure the
solvent does not cover
the spot.

Allow the plate to
develop until the solvent
is about half a
centimeter below the top
of the plate. Remove the
plate from the beaker
and immediately mark
the solvent front with a
pencil. Allow the plate to
dry.

Step 5: Visualize the spots
If there are any colored
spots, circle them lightly
with a pencil. Most
samples are not colored
and need to be visualized
with a UV lamp. Hold a
UV lamp over the plate
and circle any spots you
see. Beware! UV light is
damaging both to your
eyes and to your skin!
Make sure you are
wearing your goggles
and do not look directly
into the lamp. Protect
your skin by wearing
gloves.

If the TLC plate runs
samples which are too
concentrated, the spots
will be streaked and/or
run together. If this
happens, you will have to
start over with a more
dilute sample to spot and
run on a TLC plate.

Here's what overloaded
plates look like
compared to well-
spotted plates. The plate
on the left has a large
yellow smear; this smear
contains the same two
compounds which are
nicely resolved on the
plate next to it.

TLC Solvents Choice
When you need to determine the best solvent or mixture of solvents (a
"solvent system") to develop a TLC plate or chromatography column loaded
with an unknown mixture, vary the polarity of the solvent in several trial
runs: a process of trial and error. Carefully observe and record the results of
the chromatography in each solvent system. You will find that as you
increase the polarity of the solvent system, all the components of the
mixture move faster (and vice versa with lowering the polarity). The ideal
solvent system is simply the system that gives the best separation.
TLC elution patterns usually carry over to column chromatography elution
patterns. Since TLC is a much faster procedure than column
chromatography, TLC is often used to determine the best solvent system for
column chromatography. For instance, in determining the solvent system for
a flash chromatography procedure, the ideal system is the one that moves
the desired component of the mixture to a TLC R
f
of 0.25-0.35 and will
separate this component from its nearest neighbor by difference in TLC R
f

values of at least 0.20. Therefore a mixture is analyzed by TLC to determine
the ideal solvent(s) for a flash chromatography procedure.
Beginners often do not know where to start: What solvents should they pull
off the shelf to use to elute a TLC plate? Because of toxicity, cost, and
flammability concerns, the common solvents are hexanes (or petroleum
ethers/ligroin) and ethyl acetate (an ester). Diethyl ether can be used, but it
is very flammable and volatile. Alcohols (methanol, ethanol) can be used.
Acetic acid (a carboxylic acid) can be used, usually as a small percentage
component of the system, since it is corrosive, non-volatile, very polar, and
has irritating vapors. Acetone (a ketone) can be used. Methylene chloride or
and chloroform (halogenated hydrocarbons) are good solvents, but are toxic
and should be avoided whenever possible. If two solvents are equal in
performance and toxicity, the more volatile solvent is preferred in
chromatography because it will be easier to remove from the desired
compound after isolation from a column chromatography procedure.
Ask the lab instructor what solvents are available and advisable. Then, mix a
non-polar solvent (hexanes, a mixture of 6-carbon alkanes) with a polar
solvent (ethyl acetate or acetone) in varying percent combinations to make
solvent systems of greater and lesser polarity. The charts below should help
you in your solvent selection. You can also download this pdf chart of elution
order.

Interactions Between the Compound and the Adsorbent
The strength with which an organic compound binds to an adsorbent
depends on the strength of the following types of interactions: ion-dipole,
dipole-dipole, hydrogen bonding, dipole induced dipole, and van der Waals
forces. With silica gel, the dominant interactive forces between the
adsorbent and the materials to be separated are of the dipole-dipole type.
Highly polar molecules interact fairly strongly with the polar SiOH groups at
the surface of these adsorbents, and will tend to stick or adsorb onto the fine
particles of the adsorbent while weakly polar molecules are held less tightly.
Weakly polar molecules generally tend to move through the adsorbent more
rapidly than the polar species. Roughly, the compounds follow the elution
order given above.
The R
f
value
The retention factor, or R
f
, is defined as the distance traveled by the
compound divided by the distance traveled by the solvent.

For example, if a compound travels 2.1 cm and the solvent front travels 2.8
cm, the R
f
is 0.75:

The R
f
for a compound is a constant from one experiment to the next only if
the chromatography conditions below are also constant:
solvent system
adsorbent
thickness of the adsorbent
amount of material spotted
temperature
Since these factors are difficult to keep constant from experiment to
experiment, relative R
f
values are generally considered. "Relative R
f
" means
that the values are reported relative to a standard, or it means that you
compare the R
f
values of compounds run on the same plate at the same
time.
The larger an R
f
of a compound, the larger the distance it travels on the TLC
plate. When comparing two different compounds run under identical
chromatography conditions, the compound with the larger R
f
is less polar
because it interacts less strongly with the polar adsorbent on the TLC plate.
Conversely, if you know the structures of the compounds in a mixture, you
can predict that a compound of low polarity will have a larger R
f
value than a
polar compound run on the same plate.
The R
f
can provide corroborative evidence as to the identity of a compound.
If the identity of a compound is suspected but not yet proven, an authentic
sample of the compound, or standard, is spotted and run on a TLC plate side
by side (or on top of each other) with the compound in question. If two
substances have the same R
f
value, they are likely (but not necessarily) the
same compound. If they have different R
f
values, they are definitely different
compounds. Note that this identity check must be performed on a single
plate, because it is difficult to duplicate all the factors which influence R
f

exactly from experiment to experiment.
Troubleshooting TLC
All of the above (including the procedure page) might sound like TLC is quite
an easy procedure. But what about the first time you run a TLC, and see
spots everywhere and blurred, streaked spots? As with any technique, with
practice you get better. Examples of common problems encountered in TLC:
The compound runs as a streak rather than a spot: The
sample was overloaded. Run the TLC again after
diluting your sample. Or, your sample might just
contain many components, creating many spots
which run together and appear as a streak.
Perhaps, the experiment did not go as well as
expected.
The sample runs as a smear or a upward crescent:
Compounds which possess strongly acidic or basic
groups (amines or carboxylic acids) sometimes
show up on a TLC plate with this behavior. Add a
few drops of ammonium hydroxide (amines) or
acetic acid (carboxylic acids) to the eluting solvent
to obtain clearer plates.
The sample runs as a downward crescent: Likely, the
adsorbent was disturbed during the spotting,
causing the crescent shape.
The plate solvent front runs crookedly: Either the
adsorbent has flaked off the sides of the plate or
the sides of the plate are touching the sides of the
container (or the paper used to saturate the
container) as the plate develops. Crooked plates
make it harder to measure R
f
values accurately.
Many random spots are seen on the plate: Make sure
that you do not accidentally drop any organic
compound on the plate. If get a TLC plate and leave
it laying on your workbench as you do the
experiment, you might drop or splash an organic
compound on the plate.
You see a blur of blue spots on the plate as it develops:
Perhaps you used an ink pen instead of a pencil to
mark the origin?
No spots are seen on the plate: You might not have
spotted enough compound, perhaps because the
solution of the compound is too dilute. Try
concentrating the solution, or spot it several times
in one place, allowing the solvent to dry between
applications. Some compounds do not show up
under UV light; try another method of visualizing
the plate (such as staining or exposing to iodine
vapor). Or, perhaps you do not have any compound
because your experiment did not go as well as
planned. If the solvent level in the developing jar is
deeper than the origin (spotting line) of the TLC
plate, the solvent will dissolve the compounds into
the solvent reservoir instead of allowing them to
move up the plate by capillary action. Thus, you
will not see spots after the plate is developed.
These photos show how the yellow compound is
running into the solvent when lifted from the
developing jar.