Thin Layer Chromatography Instructions
CHM 226
O
Fe
Fe
Ferrocene
Acetylferrocene
Part 1: TLC & GC/MS – Day 1
Reference: Leonard, J., Lygo, B., Procter, G. Advanced Practical Organic Chemistry, 2nd
ed.; Nelson Thornes Ltd: United Kingdom, 2001.
For only today’s experiment, you are able to work with a partner. The objective of this
exercise is to separate a mixture of two compounds into two pure samples. This will be
accomplished on the microscale using flash chromatography, but before you can run the
flash column, you need to discover the optimal solvent system by using thin layer
chromatography (TLC). This is done by trial-and-error, using a mixture of two solvents
having different polarities. The most common polar solvent for chromatography is ethyl
acetate (EtOAc) and the most common non-polar solvent is hexanes (Hex). You will use
0%, 10%, 20%, 30%, 40% and 50% mixtures of EtOAc in Hex to find the optimal
conditions. Once you have discovered the optimal solvent combination for ferrocene
and acetylferrocene, you will use it to purify a mixture of the two compounds using flash
chromatography in the next laboratory period. The flash chromatography will be
performed using a novel method that was recently developed in the DeBoef laboratory
using prepacked silica cartridges.
Preparing the TLC Chamber
Each partner should run three TLCs using different percentages of the solvents to total
the six possibilities listed above for each pair of students. For example, one student
would use the 0%, 10%, and 20% EtOAc in Hex and the other would use the 30%, 40%,
and 50% EtOAc in Hex. Make 10 mL of each of your three mixtures and prepare a TLC
chamber for each of them. This is described in Leonard, Lygo & Procter or at these
websites:
http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html, or
http://www.youtube.com/watch?v=EUn2skAAjHk.
Thin Layer Chromatography Instructions
CHM 226
For this laboratory, TLC chambers are prepared by adding the 10mL of solvent to a
250mL beaker and using a watch glass as a cover.
Preparing the Spotting Solutions
Obtain approximately 50 mg each of ferrocene and acetylferrocene. You do not need to
weigh or record the masses. Dissolve a small portion of each of the pure compounds in
EtOAc in two separate test tubes. Mix the remainder of the compounds and dissolve a
small portion of the mixture in EtOAc in another test tube. Your TA will instruct you to
use one of these solutions to prepare a gas chromatography (GC) vial. To do this, pipet
about 0.5 mL of the solution indicated by your TA into a GC vial and label it carefully
with the compound name and your names or initials. To utilize time effectively, analyze
the samples whenever time allows instead of waiting until the end of class. Be sure to
have all three GC spectra, ferrocene, acetyleferrocene, and mixture, before you leave
class.
Spotting the TLC Plates
Before spotting the TLC plates, draw a pencil line at the bottom using a ruler. Be sure
that this line will be above the solvent line in your TLC chamber. Otherwise, your
compounds will dissolve into your solution and the TLC will need to be repeated. Draw
pencil marks on this line for pure ferrocene, the mixture, and pure acetylferrocene.
Near the top of the TLC plate, draw another pencil line with a ruler. When the solvent
reaches this line, remove the plate from the TLC chamber. To spot the TLC plates, use a
micropipette which will draw the spotting solution into itself by capillary action. Make
small spots on the pencil line of the TLC plate by using the solutions prepared earlier.
Spot the plate several times for each compound and blow air onto the plate to dry it in
between applying the spots. Develop it using one of your TLC solvents. Repeat this
procedure with your other solvent systems.
Thin Layer Chromatography Instructions
CHM 226
F = Ferrocene, M = Mixture, and A = Acetylferrocene
Visualizing the Spots
Once the solvents have run up the plates, visualize the spots (they should have moved)
using the UV boxes and circle the spots using a pencil. Be sure to use the UV boxes
because, while the spots are likely visible without it, the UV boxes will make the spots
easier to see.
Calculating Rf
Calculate the retention factor (Rf) for each of the spots using the following equation:
Rf = (distance spot moved)/(distance solvent moved).
Rf values do not have units and be aware of significant figures. Compare your Rf values
with those from the other members of the class. An optimal solvent system will provide
a good separation of the compounds and an ideal Rf = 0.3 for the lower compound.
Draw representations of your three TLC plates as well as those of your lab partner on
your datasheet.