Lab #3: Thin Layer and Column Chromatography
Summary: In this lab, you will use thin layer chromatography (TLC) to assess the ability of various solvent combinations
to separate ferrocene and acetyl ferrocene. You will then use your experimentally determined eluent to perform column
chromatography on a mixture of ferrocene and acetyl ferrocene. Finally, you will identify which compound eluted first
using a melting point analysis.
Pre-Lab:
Reading: Nichols 2.0 – 2.3A
Watch: JoVE Performing 1D Thin Layer Chromatography (9 min 47 sec)
JoVE Column Chromatography (9 min 22 sec)
JoVE Rotary Evaporation (6 min 42 sec)
JoVE Melting Point (5 min 11 sec)
Preparation: Prepare your notebook for this lab by completing sections 1-6, as detailed in the lab syllabus. Complete the
pre-lab reading and quiz!
Objectives:
• To employ a commonly used analytical technique (TLC) to assess separation conditions for a mixture of materials
• To gain an understanding of how differential polarity can be leveraged to separate materials
• To gain an understanding of a routine purification technique
Reagent and Equipment List:
Ferrocene
Acetylferrocene
Dichloromethane
Hexanes
Ethyl acetate
150 mL beaker
Watch glass
2 100 mL RBF
50 mL Erlenmeyer flask
Introduction to Thin Layer Chromatography:
Thin Layer Chromatography (TLC) is a very common and versatile analytical technique. TLC can be used to check the
progress of a reaction, to determine the purity of a compound, and even to aid in the identification of a compound or mixture.
In this experiment you will use TLC to identify an unknown over-the-counter (OTC) painkiller after selecting a developing
solvent that best differentiates the components of these OTC painkillers.
TLC consists of three steps - spotting, development, and visualization. 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 marked
with a pencil. Pencil must be used, as pen ink contains various organic compounds which will conflate your results. In this
case, our TLC plate is 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 analyte.
Development consists of placing the TLC plate into a shallow pool of a solvent (mobile phase), which then travels up the
plate by capillary action. As the solvent travels up the plate, it moves over the original spot. A competition (equilibrium) is
set up between the silica gel plate and the development solvent for the spotted analyte. 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 appear 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 the ratio of the
distance traveled by an analyte to the distance traveled by the solvent (i.e. Rf < 1).
Introduction to Column Chromatography:
While TLC is useful for quick analyses of mixtures, it is limited by the amount of material that can be used (typically
micrograms). Column chromatography uses the same essential separation principles as TLC, but on much larger scales
(milligrams to grams). Therefore, column chromatography is a technique that can be used to separate and purify materials
following a reaction whereas TLC can simply tell you how many different materials are present and roughly what they
might be.
In order to determine ideal separation conditions for column chromatography, you will first run TLC plates for a mixture of
ferrocene and acetylferrocene. Using this information, you will use the chosen solvent to separate the mixture of compounds
using column chromatography. Finally, the two compounds will be identified by running a TLC with a known or standard
at the same time as the separated compounds and comparing the Rf values for the spots. The products will be confirmed
further through melting point analysis.
CH3
C
O
Fe
Ferrocene
Fe
Acetyl Ferrocene
Procedure:
Part A: Your goal is to determine which developing solvent – hexanes, 9:1 hexanes;ethyl acetate, and 1:1 hexanes:ethyl
acetate – best differentiates ferrocene and acetyl ferrocene. You should consult your lab text and think carefully about what
criteria you will use to evaluate each solvent.
In order to identify an ideal mobile phase for the separation, prepare three developing chambers using 150 mL beakers,
watch glasses, and filter paper, as in Nichols 2.2E. Use ~5 mL of the developing solvent in each chamber. You will use
these chambers to develop your TLC plates each of which has been spotted with the standard solutions.
When handling TLC plates avoid touching the stationary phase. Hold the plate by its sides. The oils from your skin
can contaminate the TLC plate and form false “spots”. Using a pencil (ink is composed of organic dyes which will elute
along with your mixture), gently mark dots 1 cm from the bottom of each of the TLC plates. This will allow you to determine
where the compounds were originally spotted. Draw the one of the standard solutions (ferrocene or acetyl ferrocene) into a
capillary by placing one end of the capillary into the solution. Gently touch the capillary to each of the TLC plates and
quickly lift it. The tip of the capillary should only contact the TLC plate for a split second! Prolonged contact will create a
large spot, which will decrease the efficiency of the separation. Be sure to allow the plate to dry between each spotting.
Repeat this process for the second standard with a fresh capillary tube. After you finish spotting the plate, view it under the
UV light to be sure that each of your spots is concentrated enough for you to see them on the plate.
Place the plates in the developing chambers (bottom edge down), cap the chambers with the watch glasses and let them
stand in the fume hood, undisturbed. Watch as the solvent rises up the plate, it will take about 3-5 minutes for the solvent
front to get approximately 2/3 of the way up the plate. Do not allow the solvent front to get all the way to the top edge. Take
the plate out of the chamber and trace the solvent front with a pencil. Allow the TLC plate to dry in the hood.
Use the UV lamp to visualize the spots, they will show up as dark spots against a green fluorescent background. Mark the
center of the spots with a pencil. If your spots are very faint and hard to see, repeat the separation using a larger amount of
the mixture (more spots). If your spots are very large, elongated (teardrop shaped) or have “tails”, this indicates that the
plate was overloaded. Repeat with a new plate and either dilute your spotting solution or spot fewer times.
Measure and record the Rf values for each spot as shown in Nichols 2.2C. Draw a picture of every finished TLC plate in
your notebook. Be sure to label what was spotted on each dot and what solvent was used to develop the TLC.
An ideal mobile phase for column chromatography will give an Rf of ~0.3 -0.4 for your least polar compound, maximize
the separation between the two spots in the mixture, and will move the most polar spot off of the baseline of the TLC plate.
Not all these criteria can be met for every separation. Alternatively, you might choose to start with a solvent which produces
a large separation between the compounds you are separating even if the more polar compound does not move from the
baseline. Then, once the less polar compound has eluted from the column, you could switch to a more polar solvent to elute
the second, more polar compound. Which solvent mixture is most ideal for this separation?
Properly labeled TLC plate drawing
PART B:
Column Chromatography Procedure
You will need these materials during the separation. Set them aside before beginning.
• 100 mL of your selected “starting solvent”
• 2 tared 100 mL RBFs
Packing the column
Support base: Clamp the column onto a ringstand in your fume hood so that it is vertical. Carefully push a very small piece
of glass wool or cotton into the column using a long glass rod. Add just enough sand to form a layer about 3mm deep above
the glass wool. With the stopcock closed, fill the column 2/3 full with your selected “starting solvent.” Place a 125-mL
Erlenmeyer flask under the tip of the column to act as a solvent collection flask. Note: You will need only about 1g of
sand—take only what you need from the reagent bottle.
Solvent Space
Top Sand Layer, 3mm
Slurry Packed
Silica Gel
Bottom Sand Layer, 3 mm
Glass Wool Plug
Column Chromatography Apparatus
Preparing your column: Weigh out approx. 4 g of 60-200 mesh silica gel in a beaker. Prepare a slurry by slowly adding
the silica gel to 20 mL of your “starting solvent” in a 200-mL beaker. Make sure the sand layer is level and with the column
stopcock open enough to allow a slow drip, pour the slurry into the column until the column is ~½ full (see column diagram).
It helps to swirl the beaker just before pouring in the slurry. Solvent from the collection flask should be periodically recycled
into the slurry in the beaker to prevent the silica gel from drying out. While gently tapping the column with your finger,
allow the solvent to run through the column for a few minutes to allow the silica gel to settle completely, then close the
stopcock.
CAUTION: DO NOT ALLOW THE LEVEL OF THE SOLVENT TO DROP BELOW THE TOP OF THE SILICA
GEL LAYER AT ANY TIME.
Make sure there is about 1-2 inches of solvent above the column head, then add enough sand to form a layer about 3 mm
deep above the silica gel. Level the surface of the layer by gently tapping the column with one finger. DO NOT tap with a
glass rod.
Applying the sample: Add methylene chloride (CH2Cl2) to a known mass of ferrocene/acetylferrocene sample one drop at
a time while swirling until the entire solid is dissolved. The smaller the amount of CH2Cl2 you use, the better the separation
will work. Drain the solvent in the column until the top of the solvent just reaches the top sand layer. Transfer your entire
sample mixture onto the top of the column using good pipet technique while being careful not to disturb the sand.
Elution: Your goal is to collect samples of ferrocene and acetylferrocene with high purity and high mass recovery. Both of
these compounds should be collected in tared (i.e., pre-weighed round-bottomed flasks.) Any “blank” solvent from your
column should be collected in Erlenmeyer flasks and set aside until the end of the experiment.
Begin the elution process by opening the stopcock just enough to allow a steady drip of solvent from the column. Continue
to add your solvent to the column until the first colored band approaches the bottom of the silica gel. Collect this first band
in a tared 100 mL round-bottomed flask. When the first band is completely off the column, switch your mobile phase, as
discussed in our mid-lab planning session, and begin collecting the colorless solvent in a beaker. Collect the second colored
band in the second tared round-bottomed flask. You can turn the stopcock off at any time, if necessary.
Recovering the separated compounds: Carefully evaporate the solvents from your ferrocene and acetyl ferrocene samples
using the rotary evaporator. After all solvent is removed, set the flasks aside to cool and allow the compounds to crystallize.
Analysis, purification and required data: Weigh the flasks containing your dry separated products to obtain the yields of
each (in mg). Determine the melting point of the compound from the first band.
The compound from the second colored band may be sticky or gummy, if it is dry and hard enough to make a powder, take
its melting point. Scraping the residue with a metal spatula will help it to solidify. Scrape enough loose from the bottom of
the flask to use in a melting point sample. Determine the melting point (m.p.) and the weight obtained (in milligrams).
Write-Up Expectations: You will complete a Data and Calculations section which documents your solvent screening via
TLC, melting point, and yield data.
Your Discussion section should address: Which compound eluted first? How do you know? What does this suggest about
the polarity of each compound? Is this consistent with your hypothesis? How do the experimental melting points compare
to those found in a database? What was your percent recovery? If it was exceptionally low, why? Use data to support your
responses.
Finally, provide a succinct conclusion of your work.