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.

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 half a cm below the top of the beaker.
plate. 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
Rfreading.

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 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.

Column Chromatography

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

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.

• Column Chromatography Procedures Page

nteractions 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

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 Rfvalue, 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 factoVisualizing 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?

rs which influence Rf exactly from experiment to experiment.