Chromatography: refers to several related techniques for analyzing,
identifying, or separating mixtures of compounds.
The sample mixture is placed into a liquid or gas: mobile phase
The mobile phase carries the sample through a solid support:
Compounds in the sample mixture move through the stationary
phase at different rates due to different attractions for the mobile
and stationary phases
Choosing Adsorbents and Eluents
Stationary phase: Adsorbents.
Alumina is generally used for chromatography of less
Silica gel is better for compounds containing polar
least polar cyclohexane
Mobile phase: Eluents (liquid) petroleum ether
The more polar eluent = the greater eluting power: its hexane
ability to move compounds over the adsorbent toluene
Mixed eluents can be prepared by mixing low polarity ethyl acetate
and high polarity solvents and therefore creating any
eluting power needed.
most polar methanol
In TLC, capillary action allows the mobile phase (eluent) to ascend the
stationary phase (solid coated on a support plate).
A sample is applied at the bottom of a dry TLC plate.
The plate is placed in a developing chamber.
After the separation is complete, the TLC plate is called a chromatogram.
Before Development After Development
Column chromatography: technique that uses an adsorbent packed in a
glass column, and a solvent that moves down slowly through the packed
t=0 t=1 t=2 t=3 t=4
TLC vs. Column Chromatography
Adsorbent adsorbs the mixture compounds.
Eluent travels up (or down for column) over the
adsorbent, the compounds within the mixture move at
Compounds with less attraction for the adsorbent
move rapidly with the eluent.
Compounds with more attraction for the adsorbent
move slowly with the eluent.
Adsorbents vs. Eluents:
Adsorbents are typically very polar. The more polar is
a compound in the mixture, the more strongly it
adheres to the adsorbent and the more slowly it
The more polar the eluent, the more rapidly a
Retention Factor (Rf)
Definition: ratio of the distance that a compound moves to the distance that
the eluent front moves.
distance traveled by compound, mm
Rf = ————————————————————
distance traveled by eluent front, mm
Rf1 = ————
Rf2 = ————
What You Need for TLC Experiment
6 TLC Plates
5 microliter pipets (one for each
solution, do not mix them!)
4 clean test tubes
0.5 mL of “stock solution”
0.5 mL of benzophenone
0.5 mL of biphenyl
0.5 mL of benzhydrol OH
Vial with unknown (from TA)
2- Preparing the Developing Chamber
Label 3 beakers (ethyl acetate, hexane, toluene)
Pour 5-10 mL of the appropriate solvent into each beaker to moisten the
filter paper and to form a layer 3-5 mm deep
Cut a piece of filter paper to fit the beakers (used to saturate the chamber’s
atmosphere with eluent vapor)
Cover each beaker with aluminum foil
3- Spotting the TLC Plates
Obtain 3 TLC plates and label them at the top with the name of one of the 3
Mark the origin line across the plate 1 cm from the bottom, and two cross
marks on the origin line to indicate where the solution will be spotted (Use a
gentle pencil not to scratch the surface).
Place the end of the micropipet into the stock solution and allow the liquid
to rise by capillary action.
Spot the solution onto the plate by quickly and lightly touching the end of the
micropipet to the surface of the adsorbent. (diameter of the spot less than 2
Let it dry and apply a second time on one of the spots
Give 2 different
3- Developing TLC Plates
Check that the level of eluent in each chamber is
below the origin line
Place TLC plates into corresponding chambers
Cover the chambers with aluminum foil (do not
move the chamber after developing begins)
When the eluent front rises to within 1 cm of the
top of the plate, remove the plate and
immediately mark the eluent front with a pencil
Allow solvent to evaporate from the plate under a
fume hood (especially toluene: 15-20 min)
Examine the developed TLC under UV light
Use a pencil to circle spots
Whether a single application or double give
The eluent that gives better separation
Calculate Rf for each spot on the plate with the
best separation (measure from center of spot)
4- Identifying the Compounds in a Mixture
Using 2 TLC plates: 1st - stock solution, biphenyl, benzhydrol
2nd - stock solution, benzophenone
Develop the plate with the best solvent / Visualize both plates under UV
Calculate Rf for each compound
Place one of the plates in an iodine chamber and record your observations
(just for fun!)
5- Analyzing an Unknown Mixture
Obtain an unknown solution from your instructor
Spot your unknown along with the stock solution (1 TLC plate)
Develop the plate with your chosen solvent / Visualize under UV
Record the compound(s) present in the unknown
TURN IN ALL PLATES WITH YOUR REPORT (in an envelope or zip lock bag)
Column Chromatography Experiment
1- Preparing a Dry Pack Column
In a short stem 9-mm Pasteur pipet place:
A small cotton plug at its tip
Clamp the column vertically
A little bit of sand, then the alumina (height is
90 mg of the already prepared mixture:
A little bit of alumina, then sand on top
2- Eluting Ferrocene - Hexane
Label a beaker hexane, fill with hexane
Place an empty erlenmeyer flask (labeled hexane) under the column
With a Pasteur pipet, add hexane to the top of the column (allow the
liquid to flow down the side of the column, taking care not to disturb
the alumina bed)
Collect the hexane as it elutes from the column
Switch to a flask (labeled ferrocene) to collect ferrocene
3- Eluting Acetylferrocene - TBME
Label a beaker TBME(t-butyl methyl ether), fill with TBME
Place an empty erlenmeyer flask (labeled TBME) under the column
With another Pasteur pipet, add TBME to the top of the column
Collect the TBME as it elutes from the column
Switch to a flask (labeled acetylferrocene) to collect acetylferrocene
If crystals form at the tip of the column, use TMBE to rinse into the flask
After all the acetylferrocene has eluted, stop adding solvent
Observe and record the color of the solutions containing the ferrocene and
Check with TA and get his/her approval
Ferrocene in Acetylferrocene
hexane in TBME
Waste solvents are collected in the hood.
Waste from Columns:
If you have time at the end of the experiment, please try to dry out your
columns using low level pressure from the house air.
Dry column contents can be tapped out of the column into a paper towel
and disposed in the collection flask in the hood.
All glass pipets will be collected in the hood for proper disposal.
Good to know if you want to have a good grade:
Your grade will suffer if you do not submit your TLC plates. 50% of your
grade is based upon the results of your lab work and 50% upon your
answers to lab questions and the write-up of your report.
A particular slide catching your eye?
Clipping is a handy way to collect important slides you want to go back to later.