Chemistry 162
Lab 3: Thin layer chromatography of organic functional groups
Objective: Use thin layer chromatography to separate and characterize the
intermolecular forces of a mixture of benzene derivatives.
Introduction: The most widely used methods of separating components of organic
chemical mixtures involve some form of chromatography. At its simplest,
chromatography is the separation of two or more compounds by distributing
them unequally between two phases, one of which is moving (called the “mobile
phase”) and the other of which is stationary. The unequal distribution comes
about due to the different solubilities and adsorptivities these compounds have
for the two phases.
In thin layer chromatography (TLC), the stationary phase is adsorbent silica
bound to a thin flexible plastic sheet, called a TLC plate. Silica (silicon dioxide) is
considered to be a polar substance. The mobile phase is an organic solvent
system (in other words, a mixture of one or more organic solvents), which, by
capillary action, will move up the stationary phase. Solvent systems are
considered to be non-polar compared to the silica, though there are degrees of
non-polarity.
The sample mixture is applied as a small spot near one edge of the TLC plate;
this is called “spotting”. The plate is then put vertically into a solvent system
reservoir such that the spotted edge is placed down (but keeping the spots above
the level of the reservoir) and the solvent system will ascend the plate. As the
solvent system goes up, the compounds in the sample mixture will (ideally)
separate; some of the compounds should stick to the stationary phase and some
should dissolve and be carried up the plate along with the mobile phase. This
process is called “developing” the TLC plate.
To get an idea of why compounds in the mixture separate, consider a mixture
that contains both a polar and a non-polar compound. The polar compound will
favor the adsorbent silica (the stationary phase) because the silica is highly polar
(following the rule of “like dissolves like”). The non-polar compound will favor
dissolving in the non-polar solvent system and travel upward with the solvent
front. Thus, each compound will ascend the plate at different rates, with more
polar compounds tending not to rise quickly. Separation is achieved!
You will examine the TLC mobility of various benzene derivatives.
Prelab question: Predict the order of polarity of the five different benzene
derivatives, from least to most polar. Indicate your reasoning. Record this in your
notebook under the purpose and submit a copy to your instructor at the
beginning of the lab period.
Materials and methods
• 5% (w/v) solutions of benzaldehyde, benzoic acid, benzyl alcohol, methyl
benzoate and benzamide in methylene chloride
• 0.5% (v/v) glacial acetic acid in ethyl acetate
• hexane
• one 3 by 7 cm standard silica TLC plate
• capillary tubes
• one 20 mL Erlenmeyer flasks
• one 10 mL graduated cylinder
• ultraviolet viewing boxes
• developing jar
• pencil, ruler, spatula, forceps, transfer pipets
Procedure
1. Obtain a 3 cm wide TLC plate and five capillary tubes for spotting the plate.
Handle the plate by its edges, taking care not to bend the plate excessively or to
otherwise cause the silica to flake off.
2. Using a pencil (not a pen!) and a ruler, draw a straight line lightly across the
width of the plate, about 1 cm from what will be the bottom of the plate. Along
this line, leaving some space at the edges, draw lightly five evenly-spaced tick
marks; this is where you will be spotting the solutions.
3. Dip the end of a capillary tube into one of the five solutions (note which one)
and watch it fill (it won’t fill to the top). Spot the plate by lightly touching it to
the TLC plate at one of the tick marks. Note the spreading circle of moistness as
the capillary empties; make sure this spot does not exceed 2 mm in diameter (if it
seems to be getting bigger, merely lift the capillary tube off of the plate, wait for
the wet spot to dry and respot the plate at the same location – repeat this until
the tube is empty).
4. Draw an exact replica sketch of your TLC plate, labeling each spot as you
finish making it. It is an excellent visual aid, especially since you can sketch in
what the TLC plate looks like after developing.
5. Repeat step 3 with the four other solutions, taking care to use a fresh capillary
tube for each different solution.
6. A jar is used for the development chamber. Make the solvent system in the
glassware provided: a 50/50 mixture of hexane and 0.5% acetic acid in ethyl
acetate. 10 mL should be plenty of solvent; carefully pour the solvent into the jar.
The bottom of the jar should be completely covered, but the level must be
shallow enough so the spots will be completely above the level of the solvent
when the plate is in the jar.
7. Develop the plate by placing it in the jar such that it leans across the jar, facing
upward. Make sure the spots are not immersed in solvent. If that happens,
spotted material will dissolve in the pool of liquid rather than climbing up the
silica on the plate. Be careful not to slosh liquid, that will interfere with the liquid
climbing evenly across the plate and will give unreliable results. Cap the jar and
let the solvent rise (you can see an ascending solvent front as time goes on).
8. When the solvent front is about 1 cm from the top of the plate, remove the
plate from the jar and quickly mark the solvent front with the pencil. This is
critical! The line of the solvent front will disappear quickly as it evaporates, and
the distance from the spotting line to the highest point reached by the solvent is
used to calculate all the Rf values. You will have no numerical data without it!
Let the plate dry in the hood.
9. Place the dry TLC plate in the UV (ultraviolet) box; the compounds should
appear as dark purple spots. Use a pencil to outline all the spots, including any
irregularities. Make a sketch of the plate in your notebook, preserving all of the
different distances and shape irregularities. The reason for this is that the silica
will flake off the plate too readily for it to be a permanent record.
10. Generate a data table with the following headings:
TLC plate
lane number
Distance traveled
by compound (mm)
Distance traveled by
solvent front (mm)
Rf value
Give yourself enough rows to accommodate all five compounds.
11. Measurements should be made on the plate, not the drawing in the notebook.
Measure the distance from the tick mark to the solvent front in each lane and the
distance from the tick mark to the compound spot in each lane; enter the
measurements (in millimeters) in the appropriate row in the table, and also mark
it on the plate drawing.
12. Calculate the retention factor (Rf) value by dividing the distance traveled by
the compound by the distance traveled by the solvent front.
13. Using your TLC results, rank the order of polarity of the five different
benzene derivatives, from least to most polar. How did you obtain this order?
Conclusion: Were there differences between your predicted order and the
measured order? If so, what else could be going on here? Hint: Are there other
intermolecular forces involved besides dipole-dipole forces? Come up with a
new ranking based on your answer. Does this match the TLC results?
Postlab Question: Benzoic acid and benzamide are solids at room temperature,
while the rest of the benzene derivatives are liquids. Is the Rf value of a
compound (which you know is already affected by the compound’s polarity)
affected by whether it is a liquid or solid at room temperature? Using
observations from the lab, explain why or why not.