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Pre-Lab Assignment:
Complete the pre-lab cover page of the Lab-template Handout. Develop your own procedure.
Learning Goals:
In this lab you will be given a mixture containing two of the following three compounds.
O
Fluorene
OH
9-Fluorenone
9-Flourenol
You will use TLC to determine your unknowns. An example is shown below:
Before Developing
1 = known compound 1
2 = known compound 2
3 = known compound 3
4 = unknown mixuture
After Developing
unknown is a mixture
of compounds 2 & 3
solvent front
To accomplish this, you will first spot your compounds on to the TLC plate. This technique is
probably the most difficult part of this lab too much product will streak when the plates are
developed and too little will be difficult to visualize. To spot your compounds, you will use
special disposable pipets that dispense between 1 and 5 Ls. After the compounds are spotted
you must allow a few minutes for the spotting solvent to evaporate prior to developing (allowing
the solvent to migrate up the plate) the plates and visualize using UV light. The TLC plates used
in this lab contain a material that fluoresces when exposed to short wave UV light (green).
When a compound that also absorbs UV light at this wavelength is applied to the TLC plate, it
absorbs the light and quenches the background fluorescence, causing it to appear as a dark spot.
You will use hand held UV lights to visualize your TLC plates. You will also determine the
retention factor (Rf) of each known compound by dividing the distance from the origin to each
spot (in millimeters) by the distance from the origin to the solvent front. An example is shown
below
Determining Retention Factor
Note: A properly
labeled TLC Plate has
the solvent front, the
origin, and the dots
labeled. You must
properly label and
tape your TLC plate in
the observations
section of your lab
report for full credit.
solvent f ront
= 60 mm
50 mm =
origin
1
Rf =
= 0.83
Procedure:
Be sure to bring a pencil and metric ruler to lab. Prepare a developing chamber by lining the
inside of a 4 ounce vial with filter paper. What is the purpose of the filter paper? Next, add
enough methylene chloride (the elution solvent) to the vial so that the top of the liquid is about
0.5 cm from the bottom of the vial. Obtain a TLC plate from the hood and, using a pencil, gently
draw a line at about 1 cm from the bottom of the TLC plate (dotted line in the figure above).
Next, divide the line that you have drawn with short perpendicular lines to indicate where
compounds are to be spotted.
Refer to http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html for pictures of
TLC plates before, during and after development
It is important that the line on the TLC plate is above the solvent surface when it is placed into
the developing chamber.
Label (1-3) three 12x75 disposable test tubes and put them into the test tube rack. To each tube,
add a few crystals of each compound according to the table below. Tube 4, your unknown will
be provided to you and can be found in the hood. Add about 0.5 mL of methylene chloride to
each tube and shake gently to dissolve
Tube 1
fluorene
Tube 2
9-fluorenone
Tube 3
9-fluorenol
Tube 4
unknown
Using the micro pipet, spot the TLC plate with the solutions in the tubes making sure to keep the
numbering correct. Your TA will demonstrate proper spotting technique.
Use a new spotting pipette for each compound to avoid cross contamination. After spotting is
complete, allow the TLC plate to dry completely. How might the results of the TLC plate be
altered if the spots are not dry? Transfer the TLC plate to the developing chamber using forceps.
Make sure that the line on your plate is parallel to the surface of the developing liquid and
leaning against the side of the chamber. Replace the lid on the chamber, but do not tighten it.
Remove the plate when the liquid is 1cm from the top of the plate and mark the solvent front
with a pencil. Once the plate is dry, use the UV light to visualize it. CAUTION: Do not look
into the UV light, because it can damage your eyes. The spots will appear as dark dots and the
plate will glow green. Hold the light over your plate and trace the spots using a pencil.
Generally speaking, which compound, more polar or less polar, travels the furthest up the TLC
plate? Why? Measure the Rf values for each spot, using the method described above.
Post Lab: