The advantages of tlc is that the technique is
rapid, easy to perform and requires only
very small quantities of sample (as low as a
nanogram in some cases). A mixture to be
analyzed is dissolved in a volatile organic
solvent (e.g. dichloromethane or ethyl
ether). A micropipette is then dipped in the
solution and spotted near the bottom edge of
a tlc plate and the volatile solvent is allowed
to completely dry. Do not gouge into the tlc
plate when spotting the sample. Large,
diffuse spots will result in wider spots after
the tlc plate is developed. Try to keep spots
small.
The tlc plate will be put into a developing
chamber (can be a screw-top jar or beaker
covered with Al foil), which contains the
eluting solvent. The spot on the tlc plate
should be just above the surface of the
eluant (eluting solvent) when it is placed in
the developing chamber. A paper wick
(filter paper can be used) in the developing
chamber will increase the rate of elution.
The solvent will migrate up the tlc plate by
capillary action, carrying with it the various
components of the mixture. At the same
time, the components will be interacting
with the absorbent on the tlc plate. The
eluting power of the developing solvent is
directly related to the strength of adsorption
of the components on the absorbent. The
result may be a series of spots (separated
components of the mixture) in a
perpendicular line relative to the eluant.
Band width effects separation of
components in a mixture
The structure of silica gel is shown below:
OH
Si
OH
O
O
Si
O
OH
OH
Si
O Si
O
O
Si
O Si
=
O
OH
Si O Si O
O
O
O
Si O
O
What kind of compounds will interact most
strongly with silica gel?
Polar compounds will absorb more strongly
to silica gel than nonpolar compounds.
Which spots would you expect to appear
near the top of the tlc plate after
development: polar or nonpolar?
Nonpolar compounds travel through the
silica gel more quickly and appear closest
to the top of the tlc plate (closest to the
solvent front). Polar compounds
(compounds with strong dipole-dipole and
hydrogen-bonding interactions) will travel
through silica more slowly and appear near
the bottom. Tlc will separate compounds on
the basis of their polarity.
Eluants (developing solvents) that contain
more polar solvents will move components
more quickly through the absorbent. The
eluant can be adjusted to have s pots appear
in certain regions of the tlc plate.
Using a given absorbent and eluant, the rate
of movement of a compound up the tlc plate
relative to the solvent front, is a property of
that compound. The Rf value is defined as:
Rf = distance traveled by component
distance traveled by the solvent
Visualizing the spots on a tlc plate is easy if
the compounds absorb in the visible region
of the electromagnetic spectrum. For
colorless compounds, irradiating a tlc plate
with a fluorescent indicator with shortwave
uv (254 nm) light will cause most
compounds to appear as dark spots on the tlc
plate. Iodine is another commonly used
detecting agent, which works with most
compounds with unsaturated bonds. A tlc
plate can be placed in a container saturated
with iodine vapor and iodine adsorption will
cause brown spots to appear on the tlc plate.
The reaction is reversible and the brown
spots will fade once the plate is removed
from the container.
Expt. 15 – Thin Layer Chromatographic
Analysis of Drug Components
Goal: To determine the identity of an
analgesic (pain-relieving) drug prep aration
by tlc.
O
O
H3C
O
H
O
N
CH3
OH
OH
O
NH2
OH
Aspirin
Acetaminophen
O
H3C
O
CH3
CH3
N
N
O
OH
N
N
CH3
Caffeine
Salicylamide
Ibuprofen
Dissolve your drug preparation in 1:1
ethanol:dichloromethane. Using a pipette,
transfer the liquid to a vial.
Spot this solution and the standard solutions
of known drug components onto a tlc plate.
Use a separate micropipette for each
solution.
Safety Notes for Solvents
Part 1: Run the tlc plate in 200:1 ethyl
acetate:acetic acid and calculate the Rf
values for the various components. When
you remove the tlc plate from the
developing chamber, mark the solvent front
with a pencil. After the tlc plate dries,
visualize with shortwave uv light.
CAUTION: Do not look directly at the uv
lamp or shine it on your skin. Shortwave uv
(254 nm) light is in the UV-C or far uv
region of the electromagnetic spectrum.
This high-energy radiation from the sun is
screened out by the ozone layer. Less
energetic UV-B (longer wavelength)
radiation causes sunburns.
Part 2: Repeat after adding 5 drops of
methanol to your eluant.
How do your Rf values compare in parts 1
and 2?
Remember to bring a pencil and ruler for
expt. 15 to mark the starting line and solvent
front on your tlc plates.