Thin Layer Chromatography - TLC
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 (preferably 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.
TLC Adsorbent and Solvent
Usually, silica gel plates (SiO2) are used almost exclusively. (Alumina (Al2O3) can also
be used as a TLC adsorbent.) In this lab, staining (natural) was used to visualize the components.
Acetone was used as the primary solvent.
Interactions 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.
Materials
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Acetone
Baby food jars
Dried leaves
Microscope slides
Pasteur pipets
Petroleum ether
Silica gel H
Stirring rod
Preparation of TLC Plates
To make the silica gel slurry, acetone is used as the solvent .
1. Mix 20g of silica gel H with 70mL of acetone in a glass bottle large enough to hold two
microscope slides (a baby food jar works well). This amount of slurry will coat at least 20
slides. If the slurry becomes thick due to evaporation of the solvent, add a few more mL of
acetone and stir again.
2. Stir the mixture well with a glass stirring rod.
3. Place two glass slides back to back with one slightly higher than the other. Dip the slides
into the slurry and remove slowly touching the bottom of the slides to the edge of the
container to drain off excess slurry. Carefully separate the slides and set them aside to dry.
Drying will take only 10 or 20 seconds.
Preparation of Leaf Extract
4. Pulverize 2.5g of dried leaves (or material of chocice) with a mortar and pestle.
Microwave in 20 sec intervals. Fresh or frozen spinach leaves work well if dried for 48
hours in an oven or incubator at 37° C. If an oven or incubator is not available for drying
the leaves, fresh leaves may be macerated with a mortar and pestle. Maceration is more
difficult with fresh leaves but the pigment separation, #9 and #10, occurs in a similar
manner.
5. Mix the 2.5g of pulverized leaves with 3mL of acetone. Filter this mixture through filter
paper into a dark colored, easily stoppered bottle. If fresh leaves are use, mix 10mL of
acetone with 2.5g of macerated leaves. Caution: Acetone is a toxic and extremely
inflammable substance.
Placing Extract on Slide
6. Place a micropipette or pasteur pipet into the leaf extract and allow the solution to move
into the pipet by capillary action.
7. Place the pipet on the silica gel about 1mm from the bottom of the slide allowing the
solution to run onto the gel. The spot of solution will quickly dry. A second spot should be
added and allowed to dry. Continue this procedure until a dark spot is obtained.
For demonstration purposes and purely qualitative separations, large micropipets such as
pasteur pipets work quite well. For more refined separations, true micropipets should be used.
Developing the Slide
8. Place 1.5-2.0mL of chromatographic solution, a mixture of 4.5 parts of petroleum ether and
one part acetone, into the chromatojar. The chromatojar should be slightly larger than the
microscope slide and should have a tight cover (a baby food jar works well). Again,
caution: This solution is toxic and very inflammable.
9. Place the slide in the chromatojar containing the solution and quickly cover. Separation of
the pigments will occur in three or four minutes as the solvent moves up the slide.
10. When the solvent front nears the top of the silica gel, remove the slide from the
chromatojar and allow to dry. Drying will take about 10 seconds. The chromatojar and
solution may be reused many times.
11. Observe the pigments that have separated on the slide. As with paper chromatography,
carotenes will be at the top of the slide; lutein, a xanthophyll, appears next as a dark line;
the bluegreen color of chlorophyll a will be seen next; and finally near the bottom of the
slide a yellow green mixture of chlorophyll b and xanthophylls is seen.
leaf pigments
color
carotenes
golden
pheophytin
olive green
chlorophyll a
blue green
chlorophyll b
yellow green
lutein
yellow
xanthophylls
yellow