Thin Layer Chromatography - TLC

• Study Questions/Answers from the Handbook for Organic Chemistry Lab

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 page.

• TLC Procedure on this Orgchem site

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?

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 of the Using a pencil, draw a line across the plate
plate. Take care not to press so hard at the 0.5 cm mark. This is the origin: the
with the pencil that you disturb the line on which you will "spot" the plate.
adsorbent.

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 -
hold it up to the light if
necessary
touch the filled microcap
to TLC plate 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 with . . . and then draining it by

clean solvent by first touching it to a paper
 filling it . . . 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 container - The solvent will rise up the TLC
make sure the solvent is not too deep plate by capillary action. In this
photo, it is not quite halfway up the
plate.
 In this photo, it is about The solvent front is about Remove the plate from the
3/4 of the way up the plate. half a cm below the top of the beaker.
plate - it is now ready to be
removed.

quickly mark a line across Allow the solvent to

the plate at the solvent front evaporate completely from
with a pencil the plate. If the 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 spots,
lamp 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.

TLC - Retention Factor (Rf)

The retention factor, or Rf, 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 Rf is 0.75:

The Rf 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 Rf
values are generally considered. “Relative Rf” means that the values are reported relative to a
standard, or it means that you compare the Rf values of compounds run on the same plate at the
same time.

The larger an Rf 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 Rf 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 Rf value than a polar
compound run on the same plate.
The Rf 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 Rf value, they are likely (but not necessarily) the same
compound. If they have different Rf 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 Rf exactly from experiment to experiment.

In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass
(usually) column and the mobile phase, a liquid, is added to the top and flows down through the
column (by either gravity or external pressure). Column chromatography is generally used as a
purification technique: it isolates desired compounds from a mixture.

The mixture to be analyzed by column chromatrography is applied to the top of the column. The
liquid solvent (the eluent) is passed through the column by gravity or by the application of air
pressure. An equilibrium is established between the solute adsorbed on the adsorbent and the
eluting solvent flowing down through the column. Because the different components in the
mixture have different interactions with the stationary and mobile phases, they will be carried
along with the mobile phase to varying degrees and a separation will be achieved. The individual
components, or elutants, are collected as the solvent drips from the bottom of the column.

Column chromatography is separated into two categories, depending on how the solvent flows
down the column. If the solvent is allowed to flow down the column by gravity, or percolation, it
is called gravity column chromatography. If the solvent is forced down the column by positive
air pressure, it is called flash chromatography, a "state of the art" method currently used in
organic chemistry research laboratories The term "flash chromatography" was coined by
Professor W. Clark Still because it can be done in a “flash."

The Adsorbent

Silica gel (SiO2) and alumina (Al2O3) are two adsorbents commonly used by the organic chemist
for column chromatography. These adsorbents are sold in different mesh sizes, as indicated by a
number on the bottle label: “silica gel 60” or “silica gel 230-400” are a couple examples. This
number refers to the mesh of the sieve used to size the silica, specifically, the number of holes in
the mesh or sieve through which the crude silica particle mixture is passed in the manufacturing
process. If there are more holes per unit area, those holes are smaller, thus allowing only smaller
silica particles go through the sieve. The relationship is: the larger the mesh size, the smaller the
adsorbent particles.

Adsorbent particle size affects how the solvent flows through the column. Smaller particles
(higher mesh values) are used for flash chromatography, larger particles (lower mesh values) are
used for gravity chromatography. For example, 70–230 silica gel is used for gravity columns and
230–400 mesh for flash columns.
Alumina is used more frequently in column chromatography than it is in TLC. Alumina is quite
sensitive to the amount of water which is bound to it: the higher its water content, the less polar
sites it has to bind organic compounds, and thus the less “sticky” it is. This stickiness or activity
is designated as I, II, or III, with I being the most active. Alumina is usually purchased as activity
I and deactivated with water before use according to specific procedures. Alumina comes in
three forms: acidic, neutral, and basic. The neutral form of activity II or III, 150 mesh, is most
commonly employed.

Silica gel and alumina are the only column chromatography adsorbents used in the CU organic
chemistry teaching labs; please refer to the references for information on other column
chromatography adsorbents.

The Solvent

The polarity of the solvent which is passed through the column affects the relative rates at which
compounds move through the column. Polar solvents can more effectively compete with the
polar molecules of a mixture for the polar sites on the adsorbent surface and will also better
solvate the polar constituents. Consequently, a highly polar solvent will move even highly polar
molecules rapidly through the column. If a solvent is too polar, movement becomes too rapid,
and little or no separation of the components of a mixture will result. If a solvent is not polar
enough, no compounds will elute from the column. Proper choice of an eluting solvent is thus
crucial to the successful application of column chromatography as a separation technique. TLC is
generally used to determine the system for a column chromatography separation. The choice of a
solvent for the elution of compounds by column chromatography is covered in the
Chromatography Overview section.

Often a series of increasingly polar solvent systems are used to elute a column. A non-polar
solvent is first used to elute a less-polar compound. Once the less-polar compound is off the
column, a more-polar solvent is added to the column to elute the more-polar compound.

Interactions of the Compound and the Adsorbent

Compounds interact with the silica or alumina largely due to polar interactions. These
interactions are discussed in the section on TLC.

Analysis of Column Eluants

If the compounds separated in a column chromatography procedure are colored, the progress of
the separation can simply be monitored visually. More commonly, the compounds to be isolated
from column chromatography are colorless. In this case, small fractions of the eluent are
collected sequentially in labeled tubes and the composition of each fractions is analyzed by thin
layer chromatography. (Other methods of analysis are available; this is the most common method
and the one used in the organic chemistry teaching labs.)

Procedures
Column chromatography procedures are illustrated with photographs on the procedure page.