Chemistry 121
Lab 6: Thin layer chromatography of various functional groups
Prelab: Write a one page summary of you procedure. No prelab questions this week!
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 and its oxygen atoms have two lone pairs of
electrons each. 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!
In this experiment, you will examine the TLC mobility of various benzene
derivatives.
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 2 by 4 inch standard silica TLC plates
• capillary tubes
• two 10 mL graduated cylinders
• ultraviolet viewing boxes
• developing jars
• pencil, ruler, forceps, transfer pipets
Procedure
1. Obtain a 2-inch wide TLC plate and six 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, 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. Be sure to record in your notebook which tick mark corresponds to which
compound you have spotted. In fact, drawing an exact replica sketch of your
TLC plate is an excellent visual aid, especially since you can sketch in what the
TLC plate looks like after developing. It is usual to orient your sketch with the
line at the bottom.
5. Repeat step 3 with the four other solutions, taking care to use a fresh capillary
tube for each different solution.
6. Prepare the development chamber by obtaining a jar. 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. Right and cap the jar; note the level of solvent at the bottom and make
sure that when the TLC plate is put in the jar that the spots are above the level of
solvent.
7. Develop the plate by placing it in the jar such that it leans across the jar. Make
sure the spots are not immersed in solvent and make sure that the top of the
plate does not touch any of the saturated filter paper. 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 2 to 3 cm from the top of the plate, remove the
plate from the jar and quickly mark the solvent front with the pencil. 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. Make a sketch
of the plate in your notebook, preserving all of the different distances.
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. 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.
12. Calculate the retention factor (Rf) value by dividing the distance traveled by
the compound by the distance traveled by the solvent front.
Postlab Questions
1. a. Predict the order of the total intermolecular forces of the five different
benzene derivatives, from lowest to highest. Indicate why you think the order is
so.
2. Were there differences between your predicted order and the measured order?
3. If you answered yes to question 2, what else could be going on here? Come up
with a new ranking based on your answer. Does this match the TLC results?
4. 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 intermolecular forces) affected by
whether it is a liquid or solid at room temperature?
5. Using observations from the lab, explain your answer to question 4.