Title : Thin-Layer Chromatography

Objectives : To learn the technique of TLC and the visualization of colorless components.

To identify an unknown drug by a TLC comparison with standard compounds.

Introduction:

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.

Rf value = distance from origin to component spot

distance from origin to solvent front
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.
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. A balance of intermolecular forces
determines the position of equilibrium and thus the ability of the
solvent to move the solute up the plate. In other words, would the spot prefer to be stuck on the
plate or would it prefer to move along with solvent.
The balance depends upon (1) the polarity of the TLC plate (constant and high), (2) the polarity of
the development solvent (can be varied by using different solvents), and (3) the polarity of the
compounds in the spot (this varies depending upon what compounds are in the spot). For example,
if a sample consists of two components, one more polar than the other, the more polar will tend to
stick more tightly to the plate and the less polar will tend to move along more freely with the
solvent. Using a more polar development solvent would cause both to move along further. If the
approximate structures of the solutes are known, it is possible to make an educated guess as to what
solvent or mixture of solvents to use. In practice though, for a given mixture of compounds to be
analyzed, a solvent or mixture of solvents is chosen by trial and error to give the best separation. (A
caveat: the polarity argument is helpful in understanding the principles of TLC. Because most
compounds have some polarity the argument works well. For compounds having very low polarity
however, a lower-polarity solvent may be more effective in moving the solute up the plate.)
To illustrate a TLC experiment, consider the following example of the analysis of a two-component
Mixture . The polarity of molecules, solutes and solvents alike, is ordered as follows, from least to most
polar:
Alkanes (least polar), alkyl halides, alkenes, aromatic hydrocarbons, ethers, esters, ketones,
aldehydes, amines, alcohols, and carboxylic acids (most polar). Note however that many molecules
contain multiple functional groups and that the overall polarity would be determined by all of the
groups.

Experimental Procedure :
A. Spotting of the TLC plates
1. Obtain a TLC plates from your instructor. Be careful not to bend the plate excessively
since the silica adsorbent may flake off. In addition, a TLC plate should only be handled
by the edges since fingerprint contain UV active substances.
2. Set the plate down on a clean, dry surface then LIGHTLY draw a line across the plate
about 1cm from the bottom of the plate with the aid of a 2B PENCIL as shown in figure
3. Next make five 2-3mm lines, spaced about 0.8cm apart and running perpendicularly
through the lines across the bottom of the TLC plate. Be sure that the first and the last
line are about 0.8cm from the edge of the plate. These lines mark the points where the
samples will be spotted or applied to the TLC plate
4. By sharing with the people around you, spot the plate with the 5 different analgesic. Use
a separate capillary tube for each sample. First spot the acetaminophen solution, then the
caffeine, then the unknown A, then aspirin, and lastly the unknown B. it is important that
the spots be made as small as possible to avoid “tailing” when the plate is developed.
Examine the plate under the ultraviolet (UV) light to see that enough of each compound
has been applied; if not, add more.

B. Developing the TLC plate

1. Prepare a developing chamber as indicated in figure 3(ii), using a 250mL beaker as a
chamber, a half-piece of filter paper inside, and aluminium foil to cover.
2. Pour the eluent, 2 : 1 mixture of ethyl acetate : hexane, into the beaker to a depth of under
1 cm (about 15 mL). Add in about 5 drops of acetic acid into the beaker and mix. Place
the prepared TLC plate in the developing chamber, ensuring the solvent level is below
the pencil line.
3. After the solvent has risen to near the top of the plate (about 0.5cm from the top), remove
the plate and mark the solvent front with a pencil. Allow the solvent to evaporate from
the plate in the fumehood.

C. Visualization
1. The colorless compounds are visualized by illumination of the plate with an ultraviolet
(UV) lamp. Many substances, particularly aromatic compounds, will show a bright
fluorescence, which may have a characteristic color.
2. Outline the spots with a 2B pencil. The spots may also be visualized by putting the plate
in an iodine chamber for a couple minutes.
(CAUTION: DO NOT LOOK DIRECTLY AT THE UV LAMP)

D. Comparison of the unknown with reference standards

1. Sketch the plate in your notebook and calculate the R f values for each spot.
2. Determine the unknown drug based on the R f values.

Experimental Results :
Distances Moved
Lane Substances By spot (mm) By solvent (mm) Rf Value
Spotted

A Acetaminophen 0 1.6 0.198

B Caffein 0 0.8 0.099

C Unknown A 0 1.6 0.198

D Aspirin 0 2.7 0.333

E Unknown B 0 2.7 0.333

Unknown A : Acetaminophen
Unknown B : Aspirin

Discussion :
The results show that polarity of the sample is the deciding factor as to how far a component of a
sample will travel on a TLC plate. If the mobile phase is very non-polar, the non-polar components of the
sample will travel farther up the TLC plate than the polar components. If the mobile phase is very polar,
the polar components will travel farther up the TLC plate than the non-polar componentst. Also, a lot of
movement was observed when diethyl ether and dichloromethane were used as the mobile phases. In
general, this shows that the components making up the sample were more polar than non-polar, but
each had a different polarity from the other. This could be because of the eluents not being properly
covered, so they evaporated during the experiment. Also, some plates could have been left in the
mobile phase for too long and not taken out at the right time.

The results show that polarity deciding factor as to how quickly a component of a sample will travel
down a column for a collection in column chromatography. If an sample traveling through a silica gel is
less polar, the less polar component will travel down the gel quickly for collection, while the polar
component stays stationary in the gel. A more polar sample was necessary to start the more polar
component of the sample to travel down the gel for collection. TLC is very simple to use and
inexpensive. Undergraduates can be taught this technique and apply its similar principles to other
chromatographic techniques. There are little materials needed for TLC (chamber,watch glass, capillary,
plate, solvent, pencil, and UV-light). Therefore, once the best solvent is found, it can be applied to other
techniques such as High performance liquid chromatography. More than 1 compound can be separated
on a TLC plate as long as the mobile phase is preferred for each compound. The solvents for the TLC
plate can be changed easily and it is possible to use several different solvents depending on your desired
results. As stated earlier, TLC can be used to ensure purity of a compound. It is very easy to check the
purity using a UV-light. Identification of most compounds can be done simply by checking Rf literature
values. You can modify the chromatography conditions easily to increase the optimization for resolution
of a specific component.

TLC plates do not have long stationary phases. Therefore, the length of separation is limited compared
to other chromatographic techniques. Also, the detection limit is a lot higher. If you would need a lower
detection limit, one would have to use other chromatographic techniques. TLC operates as an open
system, so factors such as humidity and temperature can be consequences to the results of your
chromatogram. Based on our results, we also concluded that unknown A is Acetaminiphen and
unknown B is a Aspirin as well.

Conclusion:
In the thin layer chromatography experiment, as the mobile phase rose up on the TLC plate it dragged
the ink from the marked dot up along the TLC plate. The pigment that was closer to the marked dot was
more attracted to the stationary phase, which is the silica gel. TLC is a rapid, easy method for checking
sample purity

References :
1. Hamilton R.J., Hamilton S. "Lipid Analysis : A practical Approach" (1991) Oxford
University Press. Chapter 3. 
                                    Williamson K.L "Macroscale and Microscale Organic
Experiments" (2nd Ed.) Chapter 10. 
                                    Kirchner, J.G. "Techniques of Chemistry" (1978) 
                                    Kates, M. "Techniques of Lipidology" p231
2. https://www.academia.edu/18449142/Thin_Layer_Chromatography
3. https://uj.ac.za.libguides.com/biochemistry2a/experiment3
4. https://www.studocu.com/en/document/cleveland-state-university/organic-chemistry-lab-
i/essays/lab-report-thin-layer-chromatography/1616528/view
5. Understanding the principles of Organic Chemistry – A Laboratory Experience; Brooks/Cole
CengageLearning; Steven F. Pedersen and Arlyn M Myers; 241-245Reading assignment: Mohrig,
J.R.; Alberg, D.G.; Hofmeister, G.E.; Schatz, P.F.; Hammond, C.N.; Laboratory Techniques in
-Organic Chemistry; Freeman, New York, 2014, 255-269