Thin Layer Chromatography

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 Rf of a compound is compared
with the Rf of a known compound (preferrably 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 fluor
which fluoresces everywhere except where an organic compound is on the plate.)
The procedure for TLC, explained in words in the above paragraphs, is
illustrated with photographs on the TLC Procedure .
TLC Adsorbent
In the teaching labs at CU Boulder, we use silica gel plates (SiO2) almost
exclusively. (Alumina (Al2O3) can also be used as a TLC adsorbent.) The plates
are aluminum-backed and you can cut them to size with scissors. Our plates are
purchased ready-made from EM Sciences or from Scientific Adsorbents. The
adsorbent is impregnated with a fluor, zinc sulfide. The fluor enables most
organic compounds to be visualized when the plate is held under a UV lamp. In
some circumstances, other visualization methods are used, such as charring or
staining.
TLC Solvents or Solvent Systems
Choosing a solvent is covered on the Chromatography Overview page. The charts
at the bottom of that page are particularly useful.
Interactions of 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 Si—O bonds 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 thus generally tend to
move through the adsorbent more rapidly than the polar species. Roughly, the
compounds follow the elution order given on the Chromatography Overview page.
The Rf value
Rf is the retention factor, or how far up a plate the compound travels. See the
Rf page for more details:
• Rf's on this Orgchem site
Visualizing the Spots
If the compounds are colored, they are easy to see with the naked eye. If not, a
UV lamp is used (see the Procedure page).
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. One thing you have to be careful 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. Crookedly run plates make
it harder to measure Rf 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.
• 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. 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.
• 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?
 Thin layer chromatography

Separation of black ink on a TLC plate

Acronym TLC

Classification Chromatography

Other techniques
Thin layer
Related Agarose gel electrophoresis
SDS-PAGE

chromatography

Thin layer chromatography (TLC) is a chromatography technique used to

separate mixtures.[1] Thin layer chromatography is performed on a sheet of
glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent
material, usually silica gel, aluminium oxide, or cellulose (blotter paper). This
layer of adsorbent is known as the stationary phase.
After the sample has been applied on the plate, a solvent or solvent mixture
(known as the mobile phase) is drawn up the plate via capillary action. Because
different analytes ascend the TLC plate at different rates, separation is
achieved.[2].
Thin layer chromatography can be used to:
• Monitor the progress of a reaction
• Identify compounds present in a given substance
• Determine the purity of a substance
Specific examples of these applications include:
• determination of the components a plant contains
 • analyzing ceramides and fatty acids
• detection of pesticides or insecticides in food and water
• analyzing the dye composition of fibers in forensics, or
• assaying the radiochemical purity of radiopharmaceuticals
A number of enhancements can be made to the original method to automate the
different steps, to increase the resolution achieved with TLC and to allow more
accurate quantitation. This method is referred to as HPTLC, or "high
performance TLC".

Plate preparation
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 adsorbent layer is typically around 0.1 – 0.25 mm for analytical purposes and
around 0.5 – 2.0 mm for preparative TLC.[3]
Technique

Development of a TLC plate, a purple spot separates into a red and blue .

The process is similar to paper chromatography with the advantage of faster

runs, better separations, and the choice between different stationary phases.
Because of its simplicity and speed TLC is often used for monitoring chemical
reactions and for the qualitative analysis of reaction products.
To run a TLC, the following procedure is carried out: [4]

• A small spot of solution containing the sample is applied to a plate, about

1.5 centimeters from the bottom edge. The solvent is allowed to
completely evaporate off, otherwise a very poor or no separation will be
achieved. If a non-volatile solvent was used to apply the sample, the plate
needs to be dried in a vacuum chamber.
• A small amount of an appropriate solvent (elutant) is poured in to a glass
beaker or any other suitable transparent container (separation chamber)
 to a depth of less than 1 centimeter. A strip of filter paper is put into
the chamber, so that its bottom touches the solvent, and the paper lies
on the chamber wall and reaches almost to the top of the container. The
container is closed with a cover glass or any other lid and is left for a few
minutes to let the solvent vapors ascend the filter paper and saturate the
air in the chamber. (Failure to saturate the chamber will result in poor
separation and non-reproducible results).
• The TLC plate is then placed in the chamber so that the spot(s) of the
sample DO NOT TOUCH the surface of the elutant in the chamber, and
the lid is closed. The solvent moves up the plate by capillary action, meets
the sample mixture and carries it up the plate (elutes the sample). When
the solvent front reaches no higher than the top of the filter paper in
the chamber, the plate should be removed (continuation of the elution will
give a misleading result) and dried.
Different compounds in the sample mixture travel at different rates due to the
differences in their attraction to the stationary phase, and because of
differences in solubility in the solvent. By changing the solvent, or perhaps using
a mixture, the separation of components (measured by the Rf value) can be
adjusted. Also, the separation achieved with a TLC plate can be used to
estimate the separation of a flash chromatography column.[5]
Separation of compounds is based on the competition of the solute and the
mobile phase for binding places on the stationary phase. For instance, if normal
phase silica gel is used as the stationary phase it can be considered polar. Given
two compounds which differ in polarity, the more polar compound has a stronger
interaction with the silica and is therefore more capable to dispel the mobile
phase from the binding places. Consequently, the less polar compound moves
higher up the plate (resulting in a higher Rf value). If the mobile phase is
changed to a more polar solvent or mixture of solvents, it is more capable of
dispelling solutes from the silica binding places and all compounds on the TLC
plate will move higher up the plate. It is commonly said that "strong" solvents
(elutants) push the analyzed compounds up the plate, while "weak" elutants
barely move them. The order of strength/weakness depends on the coating
(stationary phase) of the TLC plate. For silica gel coated TLC plates, the elutant
strength increases in the following order: Perfluoroalkane (weakest), Hexane,
Pentane, Carbon tetrachloride, Benzene/Toluene, Dichloromethane, Diethyl
ether, Ethylacetate, Acetonitrile, Acetone, 2-Propanol/n-Butanol, Water,
Methanol, Triethylamine, Acetic acid, Formic acid (strongest). For C18 coated
plates the order is reverse. Practically this means that if you use a mixture of
ethyl acetate and heptane as the mobile phase, adding more ethyl acetate
results in higher Rf values for all compounds on the TLC plate. Changing the
polarity of the mobile phase will normally not result in reversed order of running
of the compounds on the TLC plate. An eluotropic series can be used as a guide
in selecting a mobile phase. If a reversed order of running of the compounds is
desired, an apolar stationary phase should be used, such as C18-functionalized
silica.
Preparative TLC
TLC can also be used on a small semi-preparative scale to separate mixtures of
up to a few hundred milligrams. The mixture is not "spotted" on the TLC plate as
dots, but rather is applied to the plate as a thin even layer horizontally to and
just above the solvent level. When developed with solvent the compounds
separate in horizontal bands rather than horizontally separated spots. Each
band (or a desired band) is scraped off the backing material. The backing
material is then extracted with a suitable solvent (e.g. DCM) and filtered to give
the isolated material upon removal of the solvent. For small-scale reactions with
easily separated products, preparative TLC can be a far more efficient in terms
of time and cost than doing column chromatography. Obviously, the whole plate
can not be chemically developed or the product will be chemically destroyed.
Thus this technique is best used with compounds that are coloured, or visible
under UV light. Alternatively, a small section of the plate can be chemically
developed e.g. cutting a section out and chemically developing it, or masking
most of the plate and exposing a small section to a chemical developer like
iodine.
Analysis
As the chemicals being separated may be colorless, several methods exist to
visualize the spots:
• Often a small amount of a fluorescent compound, usually manganese-
activated zinc silicate, is added to the adsorbent that allows the
visualization of spots under a blacklight (UV254). The adsorbent layer will
thus fluoresce light green by itself, but spots of analyte quench this
fluorescence.
• Iodine vapors are a general unspecific color reagent
• Specific color reagents exist into which the TLC plate is dipped or which
are sprayed onto the plate[6]
○ Potassium permanganate - oxidation
○ Iodine
• In the case of lipids, the chromatogram may be transferred to a PVDF
membrane and then subjected to further analysis, for example mass
spectrometry, a technique known as Far-Eastern blotting.
Once visible, the Rf value , or retention factor, of each spot can be determined
by dividing the distance traveled by the product by the total distance traveled
by the solvent (the solvent front). These values depend on the solvent used, and
the type of TLC plate, and are not physical constants. Eluent on the thin layer is
put on top of the plate
Applications
In organic chemistry, reactions are qualitatively monitored with TLC. Spots
sampled with a capillary tube are placed on the plate: a spot of starting
material, a spot from the reaction mixture, and a "co-spot" with both. A small (3
by 7 cm) TLC plate takes a couple of minutes to run. The analysis is qualitative,
and it will show if the starting material has disappeared, i.e. the reaction is
complete, if any product has appeared, and how many products are generated
(although this might be under-estimated due to co-elution). Unfortunately, TLCs
from low-temperature reactions may give misleading results, because the sample
is warmed to room temperature in the capillary, which can alter the reaction—
the warmed sample analyzed by TLC is not the same as what is in the low-
temperature flask. One such reaction is the DIBALH reduction of ester to
aldehyde.
As an example the chromatography of an extract of green leaves (for example
spinach) in 7 stages of development. Carotene elutes quickly and is only visible
until step 2. Chlorophyll A and B are halfway in the final step and lutein the
first compound staining yellow.

Step 1 Step 2 Step 3 Step 4

Step 5 Step 6 Step 7

In one study TLC has been applied in the screening of organic reactions[7] for
example in the fine-tuning of BINAP synthesis from 2-naphthol. In this method
the alcohol and catalyst solution (for instance iron(III) chloride) are place
separately on the base line, then reacted and then instantly analyzed.

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.

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 Rf 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 Rf 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 Rf
value for the red dye is:

If you could repeat this experiment under exactly the same

conditions, then the Rf values for each dye would always be the
same. For example, the Rf 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 Rf 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.

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 get, 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!)

Procedure for TLC

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:
In the teaching labs, we use a beaker with a watch glass on top.

Pour solvent into the

beaker to a depth of
just less than 0.5
cm.

To aid in the saturation

of the TLC chamber
with solvent vapors,
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.

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.
Plates will usually be cut and ready
for you when you come to lab.
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 Using a pencil, draw a line across the
of the plate. Take care not to plate at the 0.5 cm mark. This is the
press so hard with the pencil origin: the line on which you will "spot"
that you disturb the adsorbent. the plate.

It's kind of hard to see the pencil line in

the above photos, so here is a close-up of
how the plate looks after the line has been
drawn.

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.
Use a pencil and do not press down so hard
that you disturb the surface of the plate. A
close-up of a plate labeled "1 2 3" is shown
to the right.

3. Spot the TLC plate

The sample to be analyzed is added to the plate in a process called "spotting".
If the sample is not already in solution, dissolve about 1 mg in a few drops of a
volatile solvent such as hexanes, ethyl acetate, or methylene chloride. As a rule
of thumb, a concentration of "1%" or "1 gram in 100 mL" usually works well for
TLC analysis. If the sample is too concentrated, it will run as a smear or streak;
if it is not concentrated enough, you will see nothing on the plate. The "rule of
thumb" above is usually a good estimate, however, sometimes only a process trial
and error (as in, do it over) will result in well-sized, easy to read spots.
 add a few drops of . . . swirl until
solvent . . . dissolved
The solution is applied to the TLC plate with a 1µL microcap.

Not every organic lab uses microcaps to spot plates; some use drawn-
out pipets. We have chosen the microcaps from experience in working
with undergraduates; they give consistant sized spots and are
convenient. They are a bit expensive, at about $5 for 100. If you use a
new one for each spot, you could go through 10-20 in a single lab
period. Environmentally and financially, it is much better to reuse them
by rinsing between spots. Directions for rinsing and reusing microcaps
are given below.
Microcaps come in plastic vials inside red-and-white boxes. If you are opening a
new vial, you will need to take off the silver cap, remove the white styrofoam
plug, and put the silver cap back on. A small hole in the silver cap allows you to
shake out one microcap at a time. Microcaps are very tiny; the arrow points to
one, and it is hard to see in the photo.

Take a microcap and dip it into the solution of the sample to be spotted. Then,
touch the end of the microcap gently to the adsorbent on the origin in the place
which you have marked for the sample. Let all of the contents of the microcap
run onto the plate. Be careful not to disturb the coating of adsorbent.
dip the microcap into
solution - the arrow
points to the microcap,
it is tiny and hard to see
make sure it is filled touch the filled
- hold it up to the microcap to TLC plate
light if necessary to spot it - make sure
you watch to see that
all the liquid has
drained from the
microcap

do this
rinse
process
3 times!

rinse the microcap . . . and then draining it

with clean solvent by by touching it to a
first filling it . . . paper towel
If the microcap breaks or clogs, you may obtain a new one. The microcaps
should be cleaned and re-used whenever possible both because this is an
environmentally sound practice and because they are relatively expensive.

here's the TLC

plate, spotted and
ready to be
developed
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. Run until the
solvent is about half a centimeter below the top of the plate (see photos below).

place the TLC plate in the developing The solvent will rise up the TLC
container - make sure the solvent is not too plate by capillary action. In
deep this photo, it is not quite
halfway up the plate.

In this photo, it is The solvent front is Remove the plate from

about 3/4 of the way about half a cm below the beaker.
up the plate. the top of the plate - it
is now ready to be
removed.

quickly mark a line Allow the solvent to

across the plate at the evaporate completely
solvent front with a from the plate. If the
pencil spots are colored, simply
mark them with a pencil.
5. Visualize the spots
If your samples are colored, mark them before they fade by circling them
lightly with a pencil (see photo above).
Most samples are not colored and need to be visualized with a UV lamp. Hold a
UV lamp over the plate and mark any spots which you see lightly with a pencil.
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.

this is a UV here are two proper sized

lamp spots, viewed under a UV
lamp
(you would circle these while
viewing them)
The plate to the left shows three
compounds run at three different
concentrations. The middle and right
plate show reasonable spots; the left
plate is run too concentrated and the
spots are running together, making it
difficult to get a good and accurate
Rf reading.
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. The plate to the far right is a UV
visualization of the same overloaded plate

Thin Layer Chromatography

Chromatography is a word used to encompass a

range of techniques in which mixtures of pure
substances are separated into the individual
substances by using a mobile phase (usually a liquid
or gas) to push the mixture along a stationary phase
(usually a solid or liquid coated on a solid). Because
the individual substances have different molecular
structures, they interact differently with both the
stationary and mobile phases, and consequently are
"pushed" at different rates by the mobile phase. A
number of chromatographic techniques are
summarized in Table 1.
Table 1: Types of Chromatography
Type Stationary phase Mobile phase

Gas chromatography Polar or non-polar liquid Helium gas

(GC)

High Performance liquid Solid Liquid

Chromatography (HPLC)

Gel Permeation
Chromatography (GPC)

Thin Layer Solid on glass or plastic Liquid

Chromatography (TLC) plate

Thin-Layer Chromatography (TLC) is a simple and

inexpensive technique that is often used to judge
the purity of a synthesized compound or to indicate
the extent of progress of a chemical reaction. In
this technique, a small quantity of a solution of the
mixture to be analyzed is deposited as a small spot
on a TLC plate, which consists of a thin layer of
silica gel (SiO2) or alumina (Al2O3) coated on a glass
or plastic sheet. The plate constitutes the
stationary phase. The sheet is then placed in a
chamber containing a small amount of solvent, which
is the mobile phase. The solvent gradually moves up
the plate via capillary action, and it carries the
deposited substances along with it at different
rates. The desired result is that each component of
the deposited mixture is moved a different distance
up the plate by the solvent. The components then
appear as a series of spots at different locations up
the plate. Substances can be identified from their
so-called Rf values. The Rf value for a substance is
the ratio of the distance that the substance travels
to the distance that the solvent travels up the
plate. For example, an Rf value of 0.5 means that the
spot corresponding to the substance travels exactly
half as far as the solvent travels along the plate.
The Process of TLC. Performing a TLC analysis
consists of a number of steps: preparing a spotting
capillary; marking the TLC plate; spotting the TLC
plate; developing the TLC plate; drying the plate;
visualizing the substance spots, and measuring the Rf
values. We consider these steps in turn.
Preparing a spotting capillary. Glass capillaries used
for spotting TLC plates are commercially available.
However, it is occasionally necessary to make your
own capillaries. To accomplish this, light a Bunsen
burner and adjust for a medium flame. Hold a
melting point capillary in the flame until it just
begins to soften, then quickly pull the two ends of
the capillary in opposite directions. The central,
soft part of the glass will elongate and thin down to
a capillary with very small diameter. Break the two
pieces apart at the center of the thin portion to
obtain two TLC spotting capillaries.
Marking the TLC Plate. Obtain a silica gel TLC plate
that is approximately 2 cm wide and 5 cm long. Mark
the TLC plate as follows using a pencil (pencil must
be used rather than pen because inks are moved by
many developing solvents). First, LIGHTLY draw a
straight line parallel to the short dimension of the
plate, about 1 cm from one end of the plate. Don't
gouge the silica gel or make a trough with the pencil.
Second, LIGHTLY make two small marks
perpendicular to this line to divide the line into
thirds. This subdivided line will serve as a guide for
placing the substance spots, and as a point from
which to measure Rf values. Third, LIGHTLY draw a
second line parallel to the first line and about 1 cm
from the other end of the plate. When you develop
the plate, you will allow the solvent front to rise to
this second line.
Activating the TLC plate. Place your marked TLC
plate in an oven at 50-60oC for 15-20 minutes to
"activate" it. Activation involves driving off water
molecules that bond to the polar sites on the plate.
Spotting the TLC plate. Place the narrow end of one
of your capillaries into a vial containing a solution of
the substance to be analyzed, and allow the solution
to rise in the capillary; this will happen
spontaneously. Once the capillary is loaded, hold it
vertically just above the subdivided pencil line on
the plate and centered in one of the three sections.
Lower it until the narrow end of the capillary just
touches the plate right on the pencil line. You will
observe that some of the solution leaves the
capillary and deposits on the plate. Leave the
capillary in contact with the plate only briefly so
that the spot is no larger than 1 mm in diameter,
then raise the capillary. Allow the solvent to
completely evaporate from the spot (if the solvent
is water, evaporation will be slow; you can hasten it
by putting the plate in the oven for 5 minutes).
Then, if desired, make a second deposit on the same
spot with the capillary. Again allow the solvent to
completely evaporate.
Developing the TLC plate. Pour the desired
developing solvent into a small wide-mouth glass or
plastic bottle to a depth of about 4-5 mm. Using
tweezers, pick up the TLC plate at the top, which is
the end opposite where the subdivided pencil line is
drawn. Place it carefully in the developing bottle so
that it stands somewhat but not excessively tilted--
that is, the bottom of the slide should be somewhat
away from the wall of the bottle, whilc the top of
the slide rests against the wall of the bottle. It is
important that you not allow the TLC slide to tilt too
much when in the developing bottle. If the slide is
excessively tilted, solvent will not advance uniformly
along the plate and development will not take place
properly. Similarly, if the bottom of the slide is
against the wall of the bottle, solvent will advance
more rapidly up the edges of the slide than in the
middle, causing the substances to be pushed toward
the center of the slide as they move up. Leave the
slide in the chamber until solvent has advanced to
the top pencil line on the slide. Development
normally requires at least 30 minutes. When the
solvent front has advanced to the top pencil line, use
the tweezers to withdraw the slide from the
chamber.
Drying the Plate. Place the plate flat on a clean dry
surface and allow the solvent to completely
evaporate. If the solvent is not highly volatile, this
can be facilitated by placing the slide on a flat
surface in an oven at a temperature of 50-60 oC
(higher temperatures will melt the plastic substrate
material). When the plate is completely dry, it is
ready for visualization.
Visualization of the TLC Plate. If the substances
being separated are colored, the spots can be seen
without any further effort. Using a pencil, draw a
boundary around each spot that matches the shape
of the spot. Many substances are colorless (white)
and do not show up on the white silica gel unless
steps are taken to make them visible. There are a
number of techniques for doing this. First is the
technique of iodination. The dry plate is placed in a
chamber containing a few crystals of iodine. The
iodine vapor in the chamber oxidizes the substances
in the various spots, making them visible to the eye.
Once the spots are visible, they may be outlined
with a pencil before the iodine coloration fades.
Second is the ninhydrin technique, which is
particularly effective for visualizing amino acid
spots. In this method, a solution containing 0.2%
ninhydrin in ethanol is sprayed on the dry plate.
Alternately, the plate can be dipped in ninhydrin
solution. In contact with an amino acid, ninhydrin
displays a purple coloration that is easily seen. It
usually takes a few minutes for this color to develop,
so after the plate is sprayed, it is allowed to sit for
several minutes. Placing it in a 50 oC oven will hasten
the appearance of the purple color. Once this
appears, the spots may be circled in pencil to
permanently mark their positions. NOTE:
NINHYDRIN STAINS THE SKIN PURPLE, TOO,
DUE TO THE PRESENCE OF PROTEIN (AMINO
ACIDS!). TO AVOID THIS, WEAR GLOVES WHEN
HANDLING IT. Third, one may use TLC plates that
have been loaded with a fluorescent substance that
is uniformly distributed in the silica gel. Substances
moving up the plate block this fluorescence at their
locations. When the dried plate is viewed using a
special UV light, the substance spots are visible for
their lack of fluorescence on an otherwise uniformly
fluorescing field. The spots may be outlined with
pencil while being viewed in the light.
Measurement of Rf. The distance between the 2
horizontal pencil lines is the distance of solvent
advance. The distance from the bottom pencil line to
the center of a substance spot is the distance of
advance of the substance. The ratio of substance
advance distance to solvent advance distance is the
Rf value for the substance. The figure shows a
developed plate for a mixture consisting of 4
components with Rf values of about 0.05, 0.2, 0.5,
and 0.9

TLC of Inks. Before attempting to apply TLC to the

challenging problem of separating and identifying
amino acids, it is advisable to learn and practice the
technique by applying it to mixtures that are easily
visualized and separated. Inks provide an ideal
practice vehicle for TLC because they normally
contain several colored components that separate
nicely in common solvents such as ethanol, acetone,
or chloroform. Spotting the plate is also easy: it may
be done simply by making a VERY small mark on the
plate with the tip of a pen, just above the pencil line
drawn across the bottom edge of the plate. Inks are
of different types and colors, of course. Some are
washable (water-soluble), others are permanent.
Different types require different solvents for
development. Common sources of ink are ball point
pens, felt-tip markers, and roller ball pens. Bottled
ink is still available for people using fountain pens.
Particularly good ink sources are bottled inks by
Parker, Sheaffer, and Mont Blanc; Sharpie marker
inks (black, red, blue, orange, brown, yellow, green);
Marks-a-Lot Stay sharp markers; and Sanford
calligraphy pens.You should select inks from at least
2 different sources available in the lab and should
find a solvent or mixture of solvents that separates
each ink into its component colors.
Experimental Procedure. You will be assigned two ink
samples to examine. Obtain four TLC plates, four
TLC spotting capillaries, and 3 developing tanks
(snap-top plastic vials). Mark your TLC plates with a
narrow pencil line about 1cm from the bottom.
Prepare spotting solutions of your ink samples in 1-
dram vials by diluting 1 drop of ink with 9 drops of
ethanol (this is called a 1-to-10 dilution). Insert one
end of a spotting capillary into the first ink solution.
You should see the ink solution rise in the capillary.
Withdraw the capillary when the liquid has risen to a
height of 1-2 cm. Hold the capillary vertically and
briefly touch the filled end to the pencil line, about
5 mm from the left edge of the TLC plate. Liquid
should flow from the capillary to the plate to form a
spot. THIS HAPPENS RAPIDLY. If possible, lift the
capillary before the spot gets any bigger than 1 mm
in diameter. Allow the ethanol to evaporate. Touch
the capillary to a Kimwipe to empty it of ink solution.
Use the other end of the same capillary to draw up
your second ink solution, and spot the plate about 5
mm from the right edge. Discard the capillary in the
broken glass receptacle. Allow the ethanol to
evaporate from the plate.
Into your first developing tank, pour ethanol to a
depth of about 5 mm. Using tweezers to hold the
top end of the spotted plate, lower the plate into
the tank. Allow the bottom of the plate to rest on
the bottom of the tank, with the top of the plate
leaning against a tank sidewall. NOTE: IT IS VERY
IMPORTANT THAT THE START LINE AND SPOTS
BE ABOVE THE LEVEL OF SOLVENT. Close the
tank and monitor the movement of solvent up the
plate. When solvent has advanced to the top pencil
line, remove the plate from the tank, and allow all
solvent to evaporate. Use a pencil to outline each
observed spot on the plate, preserving the shape of
the spot.
If ethanol did not effect a separation of the ink
into components, try another solvent. If you found
that the ink moved along with or close to the solvent
front, try a less polar solvent. If you found that the
ink did not move at all with ethanol, try a more polar
solvent (e.g., methanol). Your goal is to cleanly
separate each of your ink samples into its
constituents, each constituent producing a single
TLC spot. Be aware that some inks contain only one
component!
TLC of Amino Acids. TLC of amino acids is more
difficult than TLC of inks, because amino acids are
colorless. Therefore, not only can you not monitor
their progress up the plate, but you cannot see the
spots with the naked eye once the plate is fully
developed and dried. To see the spots, it is
necessary to use either the ninhydrin or the black-
light visualization techniques. Of course, the latter
works only if you use fluorescent TLC plates, which
are available in the lab. Until you see the spots, you
will not know whether or not a chosen solvent
system has been effective in moving an amino acid
or in separating a mixture. Therefore the process of
finding an effective solvent system can be long and
painstaking. For this reason, we will specify the
solvent system that you are to use. As points of
general information, amino acids are quite polar and
tend to move on silica gel plates with polar solvents.
They have Rf values close to 1 when water or
concentrated ammonia is used as the developing
solvent, probably because of their high solubility in
water. Diluting a polar solvent with a less polar one
results in smaller Rf values, roughly in proportion to
the amount of less polar solvent used, Thus, alanine,
glycine, threonine, and proline all have Rf values of
around 0.60 when developed with a 50/50 mixture
of water and n-propanol, and around 0.40 when
developed with a 30/70 mixture of concentrated
NH3 and n-propanol. The following procedure
assumes the use of 50/50 water/n-propanol as the
developing solvent, but you are free to try other
polar/non-polar combinations.
Experimental Procedure. In the hood, prepare 10 mL
of a mixture consisting of 50% 1-propanol and 50%
water by volume, and pour about half of this into a
clean developing tank. Make sure that the level of
liquid in the tank is no higher than 5 mm, and close
the lid. In a 1-dram vial, prepare a solution of about
0.001 g of your amino acid in 0.2 mL of water.
Dissolve the acid, then draw some solution up in a
spotting capillary and double-spot a properly marked
and activated TLC plate. Allow the plate to dry for 5
minutes, then use tweezers to carefully lower the
plate into the developing tank so that its bottom is
submerged in the developing solvent. NOTE: IT IS
VERY IMPORTANT THAT THE START LINE AND
SPOTS BE ABOVE THE LEVEL OF SOLVENT. Close
the lid, and allow the plate to develop until solvent
has risen to the pencil line at the top of the plate.
Remove the plate from the tank and place it in an
oven at 50 oC to dry. When the plate is dry, visualize
it using ninhydrin spray or iodination. Circle the
amino acid spots with pencil, and calculate Rf values.
Compare the measured Rf values with the values
posted for the amino acids.
On this basis, you can narrow down the identity of
your amino acid. In combination with other data that
you obtain, this information will help you
unambiguously identify your amino acid. Suppose
that your amino acid has an Rf value similar to that
of, say, alanine. You should then prepare a small
amount of alanine solution and spot it alongside your
amino acid on a new TLC plate. Develop, dry, and
visualize the plate to confirm that your amino acid
indeed has exactly the same Rf value as alanine, and
that the spot is the same shape and color.
Finally, it is very important to be observant of detail
in doing TLC. In addition to the Rf value for a
substance, the shape of the spot produced by a
particular developing solvent and the shade of color
produced by iodine or ninhydrin can be
characteristic of the substance. Please note all of
these things. For example, when alanine, glycine,
threonine, and proline are spotted side-by-side on a
plate and developed with 70% n-propanol/30% conc
NH3, the following observations can be made:

Amino Acid Solvent Spot Color Spot

after with
Iodination Ninhydrin

alanine 30/70 conc white on purple

NH3/n- brown bkgrnd
propanol

alanine 50/50 white on purple

water/n- brown bkgrnd
propanol

glycine 30/70 conc white on pink

NH3/n- brown bkgrnd
propanol
glycine 50/50 white on pink
water/n- brown bkgrnd
propanol

threonine 30/70 conc white on purple

NH3/n- brown bkgrnd
propanol

threonine 50/50 white on purple

water/n- brown bkgrnd
propanol

proline 30/70 conc dark brown on yellow with

NH3/n- brown bkgrnd pink border
propanol

proline 50/50 white on yellow with

water/n- brown bkgrnd pink border
propanol

Problems in TLC.
Over-large Spots. Sample spots made using TLC
capillaries should be no larger than 1-2 mm in
diameter, because component spots in the developed
plate will be no smaller than, and will usually be
larger than, the size of the initial spot. If the initial
spot is larger than 2 mm in diameter, then
components with similar Rf values may not be
resolved because their spots will be so large that
they will overlap considerably and may appear to be
one large spot. Small initial spots, on the other hand,
maximize the potential of complete separation of
components.
Uneven Advance of Solvent Front. A common
problem in TLC is uneven advance of solvent along
the plate. Instead of a straight line, the solvent
front may appear to bow either up or down in the
center. Uneven advance of solvent leads to uneven
advance of substance spots, and inaccurate Rf values
result. A frequent cause of uneven solvent advance
is the use of a developing chamber that does not
have a flat bottom. Glass bottles usually have
bottoms that curve upward from the edges to the
center. If the bottom of the TLC plate is placed on
this curved surface, the shape of the solvent
advance line may mirror the shape of the container
bottom. It is therefore important to use flat-
bottomed developing tanks in TLC. A bowed solvent
front may also result if too little developing solvent
is placed in the chamber; if the plate is cut
improperly, so that the sides are not exactly
perpendicular to the bottom edge; and if the slide is
excessively tilted in the chamber. Care in choosing
and using a developing chamber is the best defense
against curved solvent fronts.
Water is seldom used as a developing solvent
because it has a tendency to produce a dramatically
curved front. This may be due to its unusually high
surface tension.
Streaking. Sometimes a substance will move along a
TLC plate as a long streak, rather than as a single
discrete spot. This is the result of spotting the
plate with too much substance, more than the
moving solvent can handle. The solvent moves as
much substance as it can, but a substantial amount
of substance is left behind. The substance is
dragged along by the solvent leaving a trail of
substance that may sometimes span the entire
distance between the starting line and the solvent
front. Streaking can be eliminated by systematically
diluting the spotting solution until development and
visualization show the substances moving as single
spots, rather than elongated streaks.