The Technique of Thin-Layer Chromatography

Introduction: Chromatography is the process in which two or more compounds are separated through
the cause molecular interaction of different with two phases. These interactions can be pairs of liquid
and a liquid, a solid and a liquid, a gas and a solid, etc. This process can be shown in the laboratory
through the use of cellulose paper as the stationary phase and a propanol and water mixture and the
moving phase liquid. The paper would have different ink on the edge and by dipping the edge of the
paper into the mobile mixture you can observe the separation of each substance. In the method of Thin-
Layer Chromatography this principle can still be applied. It is a common, inexpensive, practical technique
used to do organic experiments and is can detect small sample sizes. A polar solid absorbent and single
or combination solvents would be used as the stationary and mobile phases respectively. In todays
experiment, we be practicing the technique of TLC.

Materials:

Aspirin Molecular Formula:

C9H8O4
Formula Weight:
180.16
Acetaminophen Molecular Formula:
C8H9NO2
Formula Weight:
151.16
Ibuprofen Molecular Formula:
C13H18O2
Molecular Weight:
206.285 g/mol
Caffeine Molecular Formula:
C8H10N4O2
Formula Weight:
194.19

Pencil
Silicia gel
TLC plates
Spotting Capillary
Tweezers
UV light
Pastuer Pipette
Beaker

Procedure:
The use of commercially available TLC plates, poly(ethylene terephthalate) (Mylar) sheets coated with
silica gel using polyacrylic acid as a binder, is highly recommended; these fluoresce under ultraviolet
(UV) light.

1. The TLC plates must be handled gently and by the edge, or the 100-mm-thick coating of silica
gel can be easily scratched off or contamination of the surface can occur.
2. With a pencil, lightly draw a faint line 1 cm from the end and then three or four short hash
marks to guide spotting.
3. Lightly write identifying letters at the top of the plate to keep track of the placement of the
compound spots (Fig. 8.6). Note that a pencil is always used to mark TLC plates because the
graphite (carbon) is inert.
4. If ink is used to mark the plate, it will chromatograph just as any other organic compound,
interfering with the samples and giving flawed results.
5. Once the sample is prepared, a spotting capillary must be used to add the sample to the plate.
Spotting capillaries can be made by drawing out open-end melting point tubes
6. The bore of these capillaries should be so small that once a liquid is drawn into them, it will not
flow out to form a drop.
7. Practice spotting Citral from the previous experiment onto an unmarked TLC plate. Dip the
capillary into the solvent and let a 23 cm column of solvent flow into it by capillary action.
8. Do this by holding the capillary vertically over the coated side of the plate, and lower the
pipette until the tip just touches the adsorbent.
9. Only then will liquid flow onto the plate; quickly withdraw the capillary when the spot is about 1
mm in diameter. The center of the letter of on this page is more than 1 mm in diameter. The
solvent will evaporate quickly, leaving your mixture behind on the plate.
10. You may have to spot the plate a couple of times in the same place to ensure that sufficient
material is present; do not spot too much sample because this will lead to a poor separation. It
is extremely important that the spots be as small as possible. If the spot is large, then two or
more spots of a sample may overlap on the TLC plate, thus causing erroneous conclusions about
the separation and/or the samples purity or content.
11. Examine the plate under UV light to see the components as dark spots against a bright green-
blue background.
12. Outline the spots with a pencil. The spots can also be visualized by putting the plate in an iodine
chamber made by placing a few crystals of iodine in the bottom of a capped 4-oz jar.
13. Calculate the Rf values for the spots and identify the components in the unknown.

Before proceeding, practice the TLC spotting technique described earlier. Following that procedure,
draw a light pencil line about 1 cm from the end of a chromatographic plate.

1. On this line, spot aspirin, acetaminophen, ibuprofen, and caffeine, which are available as
reference standards. Use a separate capillary for each standard (or rinse the capillary carefully
before reusing).
2. Make each spot as small as possible, preferably less than 0.5 mm in diameter. Examine the plate
under UV light to see that enough of each compound has been applied; if not, add more. On a
separate plate, run the unknown and one or more of the standards.
 3. To the developing jar or beaker, add 4 mL of the mobile phase, a mixture of 95% ethyl acetate
and 5% acetic acid. Insert the spotted TLC plates with tweezers.
4. After the solvent has risen nearly to the top of the plate, remove the plate from the developing
chamber, mark the solvent front with a pencil, and allow the solvent to dry.
5. Examine the plate under UV light to see the components as dark spots against a bright green-
blue background.
6. Outline the spots with a pencil. The spots can also be visualized by putting the plate in an iodine
chamber made by placing a few crystals of iodine in the bottom of a capped 4-oz jar.
7. Calculate the Rf values for the spots and identify the components in the unknown.

Cleaning Up. Solvents should be placed in the organic solvents waste container; dry, used
chromatographic plates can be discarded in the nonhazardous solid waste container.

Conclusion:

The experiment was successful unfortunately the experimental data was lost. The data was put into my
bag and got ruined. The Rf calculation was unable to be done but what I can tell from my observation of
the experiment was that my Citral sample was very pure as the TLC of it was absorbed in almost straight
dotted line though I could not gather anything from the other TLC. I know in the future this would not be
acceptable and therefore this experiment was not very successful and non-conclusive.

References:

Macroscale and Microscale Organic Experiments; Kenneth L.Williamson ; Katherine M. Masters

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