Thin Layer Chromatography.
Thin layer chromatography, or TLC, is a method for analyzing mixtures by separating the
compounds in the mixture. TLC can be used to help determine the number of components in a
mixture, the identity of compounds, and the purity of a compound. By observing the appearance of
a product or the disappearance of a reactant, it can also be used to monitor the progress of a
reaction. TLC is a sensitive technique - microgram (0.000001 g) quantities can be analyzed by TLC
- and it takes little time for an analysis (about 5-10 minutes).
TLC consists of three steps - spotting, development, and visualization. Photographs of each step are
shown on the course website. First the sample to be analyzed is dissolved in a volatile (easily
evaporated) solvent to produce a very dilute (about 1%) solution. Spotting consists of using a micro
pipet to transfer a small amount of this dilute solution to one end of a TLC plate, in this case a thin
layer of powdered silica gel that has been coated onto a plastic sheet. The spotting solvent quickly
evaporates and leaves behind a small spot of the material.
Development consists of placing the bottom of the TLC plate into a shallow pool of a development
solvent, which then travels up the plate by capillary action. As the solvent travels up the plate, it
moves over the original spot. A competition is set up between the silica gel plate and the
development solvent for the spotted material. The very polar silica gel tries to hold the spot in its
original place and the solvent tries to move the spot along with it as it travels up the plate. The
outcome depends upon a balance among three polarities - that of the plate, the development solvent
and the spot material. If the development solvent is polar enough, the spot will move some distance
from its original location. Different components in the original spot, having different polarities, will
move different distances from the original spot location and show up as separate spots. When the
solvent has traveled almost to the top of the plate, the plate is removed, the solvent front marked
with a pencil, and the solvent allowed to evaporate.
Visualization of colored compounds is simple – the spots can be directly observed after
development. Because most compounds are colorless however, a visualization method is needed.
The silica gel on the TLC plate is impregnated with a fluorescent material that glows under
ultraviolet (UV) light. A spot will interfere with the fluorescence and appear as a dark spot on a
glowing background. While under the UV light, the spots can be outlined with a pencil to mark their
locations. A second method of visualization is accomplished by placing the plate into iodine vapors
for a few minutes. Most organic compounds will form a dark-colored complex with iodine. It is
good practice to use at least two visualization techniques in case a compound does not show up with
one particular method.
The Rf value is used to quantify the movement of the materials along the plate. Rf is equal to the
distance traveled by the substance divided by the distance traveled by the solvent. Its value is
always between zero and one. A TLC analysis might be summarized something like, "Using a silica
gel plate and ethyl acetate as the development solvent, unknown mixture X showed three spots
having Rf's of 0.12, 0.25, and 0.87". Comparing these Rf's with the Rf's of known compounds might
enable a tentative identification to be made. Note that observing three spots means only that there
are at least three components in the mixture. Some components may have such similar polarities
that they appear under one spot after development.
1
, Rf = Y/X (always ≤ 1)
If a development solvent of too high a polarity is used, all components in the mixture will move
along with the solvent and no separation will be observed (Rf’s will be too large). If the solvent is
of too low a polarity the components will not move enough, and again separation will not occur
(Rf’s will be too small). In practice, different solvents or mixtures of solvents are tried until a good
separation is observed. Typically an effective solvent is one that gives Rf's in the range of 0.3 - 0.7.
Note that the spotting solvent is simply used as a vehicle to transfer the material to be analyzed to
the TLC plate. Once the transfer is made the spotting solvent evaporates. It has no effect on the
separation. It is the development solvent that effects the separation.
What’s going on at the molecular level during development? There are three components in TLC:
(1) the TLC plate (stationary phase), the development solvent (mobile phase), and the sample to be
analyzed (solute). In our experiment the TLC plate consists of a thin plastic sheet covered with a
thin layer of silica gel, a portion of the structure of which is shown below.
OH OH OH
Si O Si O Si O
O O O
Si Si Si O
O O
Silica gel consists of a three-dimensional network of thousands of alternating silicon and oxygen
bonds, with O-H groups on the outside surface. Silica gel is simply very finely ground very pure
sand. It should be noted that silica gel is highly polar and is capable of hydrogen bonding.
Consider the side-on view of the development of a TLC plate below. As the solvent travels up the
plate, over the spot, an equilibrium is set up, as development solvent competes with the TLC plate
for the solute. The silica gel binds to the solute and the development solvent tries to dissolve it
away, carrying the solute(s) along as the solvent travels up the plate.
2
Thin layer chromatography, or TLC, is a method for analyzing mixtures by separating the
compounds in the mixture. TLC can be used to help determine the number of components in a
mixture, the identity of compounds, and the purity of a compound. By observing the appearance of
a product or the disappearance of a reactant, it can also be used to monitor the progress of a
reaction. TLC is a sensitive technique - microgram (0.000001 g) quantities can be analyzed by TLC
- and it takes little time for an analysis (about 5-10 minutes).
TLC consists of three steps - spotting, development, and visualization. Photographs of each step are
shown on the course website. First the sample to be analyzed is dissolved in a volatile (easily
evaporated) solvent to produce a very dilute (about 1%) solution. Spotting consists of using a micro
pipet to transfer a small amount of this dilute solution to one end of a TLC plate, in this case a thin
layer of powdered silica gel that has been coated onto a plastic sheet. The spotting solvent quickly
evaporates and leaves behind a small spot of the material.
Development consists of placing the bottom of the TLC plate into a shallow pool of a development
solvent, which then travels up the plate by capillary action. As the solvent travels up the plate, it
moves over the original spot. A competition is set up between the silica gel plate and the
development solvent for the spotted material. The very polar silica gel tries to hold the spot in its
original place and the solvent tries to move the spot along with it as it travels up the plate. The
outcome depends upon a balance among three polarities - that of the plate, the development solvent
and the spot material. If the development solvent is polar enough, the spot will move some distance
from its original location. Different components in the original spot, having different polarities, will
move different distances from the original spot location and show up as separate spots. When the
solvent has traveled almost to the top of the plate, the plate is removed, the solvent front marked
with a pencil, and the solvent allowed to evaporate.
Visualization of colored compounds is simple – the spots can be directly observed after
development. Because most compounds are colorless however, a visualization method is needed.
The silica gel on the TLC plate is impregnated with a fluorescent material that glows under
ultraviolet (UV) light. A spot will interfere with the fluorescence and appear as a dark spot on a
glowing background. While under the UV light, the spots can be outlined with a pencil to mark their
locations. A second method of visualization is accomplished by placing the plate into iodine vapors
for a few minutes. Most organic compounds will form a dark-colored complex with iodine. It is
good practice to use at least two visualization techniques in case a compound does not show up with
one particular method.
The Rf value is used to quantify the movement of the materials along the plate. Rf is equal to the
distance traveled by the substance divided by the distance traveled by the solvent. Its value is
always between zero and one. A TLC analysis might be summarized something like, "Using a silica
gel plate and ethyl acetate as the development solvent, unknown mixture X showed three spots
having Rf's of 0.12, 0.25, and 0.87". Comparing these Rf's with the Rf's of known compounds might
enable a tentative identification to be made. Note that observing three spots means only that there
are at least three components in the mixture. Some components may have such similar polarities
that they appear under one spot after development.
1
, Rf = Y/X (always ≤ 1)
If a development solvent of too high a polarity is used, all components in the mixture will move
along with the solvent and no separation will be observed (Rf’s will be too large). If the solvent is
of too low a polarity the components will not move enough, and again separation will not occur
(Rf’s will be too small). In practice, different solvents or mixtures of solvents are tried until a good
separation is observed. Typically an effective solvent is one that gives Rf's in the range of 0.3 - 0.7.
Note that the spotting solvent is simply used as a vehicle to transfer the material to be analyzed to
the TLC plate. Once the transfer is made the spotting solvent evaporates. It has no effect on the
separation. It is the development solvent that effects the separation.
What’s going on at the molecular level during development? There are three components in TLC:
(1) the TLC plate (stationary phase), the development solvent (mobile phase), and the sample to be
analyzed (solute). In our experiment the TLC plate consists of a thin plastic sheet covered with a
thin layer of silica gel, a portion of the structure of which is shown below.
OH OH OH
Si O Si O Si O
O O O
Si Si Si O
O O
Silica gel consists of a three-dimensional network of thousands of alternating silicon and oxygen
bonds, with O-H groups on the outside surface. Silica gel is simply very finely ground very pure
sand. It should be noted that silica gel is highly polar and is capable of hydrogen bonding.
Consider the side-on view of the development of a TLC plate below. As the solvent travels up the
plate, over the spot, an equilibrium is set up, as development solvent competes with the TLC plate
for the solute. The silica gel binds to the solute and the development solvent tries to dissolve it
away, carrying the solute(s) along as the solvent travels up the plate.
2
