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Thin Layer Chromatography

Pre-Lab Assignment:
Complete the pre-lab cover page of the Lab-template Handout. Develop your own procedure.
Learning Goals:

Understand the principles and applications of thin layer chromatography.


You will be using TLC throughout the semester. It is important that you thoroughly
understand the concepts of TLC.
Introduction:
Thin layer chromatography (TLC) is a separation technique commonly used to monitor reaction
progress, develop large-scale separation schemes, and identify unknown compounds. TLC plates
consist of a glass, plastic, or metal sheet coated with a thin layer of porous silica gel (SiO2) onto
which a compound is applied (spotted). TLC plates are developed by partially immersing in a
chamber containing a solvent mixture. As the solvent is pulled up the plate by capillary action,
compounds are carried along (migrate or elute) - desorbing and absorbing to the silica gel at
different rates to afford separation. In general polar compounds migrate the least and non-polar
compounds migrate the most in this type of chromatography. Varying the elution solvent polarity
will increase or decrease the degree of compound migration. A more polar solvent will interact
more strongly with polar portions of dissolved molecules, decreasing their availability. This
decreased availability of the polar regions of the molecules decreases the amount of interaction
the molecules can have with the polar silica gel on the TLC plate itself. The result is that polar
solvents tend to make molecules travel further up the plate by decreasing the amount of
interaction (and thus resistance) the plate has with the molecules. The opposite is true of less
polar solvents, which dont interact with the polar portions of molecules, allowing greater
interaction between the molecule and the TLC plate. The compounds separated on the TLC
plates can be made visible by a variety of means including using UV light. Several excellent
websites that give information on TLC are given below:
General theory:
http://en.wikipedia.org/wiki/Chromatography
Specific TLC techniques:
http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html
http://www.usm.maine.edu/~newton/TANES/TLC.HTML
Journals:
Thin-layer chromatography: The "eyes" of the organic chemist
Hamilton Dickson, Kevin W Kittredge, Arlyne M Sarquis. Journal of Chemical Education.
Easton: Jul 2004. Vol. 81, Iss. 7; p. 1023
What factors affect the separation of substances using thin-layer chromatography?
John J Nash, Jeanne A Meyer, Barbara Everson. Journal of Chemical Education. Easton: Mar
2001. Vol. 78, Iss. 3; p. 364

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 =

distance from origin to red spot = 50 mm


distance from origin to solvent front = 60 mm

= 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:

Include the material required in the Lab Expectations handout.


Tape a properly labeled TLC plate to the observations section of your lab report.
Determine and list the Rf values for known compounds 1 through 3.
Determine and list the Rf values for your unknown sample: Identify the components of
the unknown mixture.

Answer the following questions separate from your conclusions.


1. Contrast liquid/liquid extraction and TLC.
2. What are the non-polar and polar phases in TLC?
3. Suppose you were visualizing a TLC plate and all the spots were at the solvent
front. How could you remedy this problem?
4. How could TLC be used to analyze the progress of a chemical reaction?
5. Name and explain two other types of analytical techniques used in organic
chemistry.

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