COLUMN CHROMATOGRAPHY: SEPARATION OF A MIXTURE
OF FLUORENE AND FLUORENONE (8/3/05)
We have already seen how mixtures of compounds may be separated by thin-layer
chromatography (TLC). However, TLC works only on a very small (milligram) scale.
When larger amounts (gram-scale) of material must be separated, column
chromatography is used. Column chromatography works on the same principle as TLC
except that the adsorbent is used in larger quantities and in a column. Typically, the
adsorbent, usually either alumina (Al2O3) or silica gel (SiO2.xH2O), is packed in a glass
column or buret. The mixture to be separated is dissolved in a small volume of an
appropriate solvent and applied to the top of the column. Then an eluting solvent (the
eluent) is passed through the column. As the eluent passes down the column, the
components of the mixture, which were initially adsorbed onto the stationary phase (the
adsorbent), begin to move down the column. The components move down the column at
different rates depending upon how strongly they are attracted to the adsorbent, the more
weakly a component is adsorbed, the faster it is eluted from the column. Thus the
components move down the column in bands or zones, which are collected as different
fractions as they exit the bottom of the column. If the components are colored, the
progress of the elution can be followed by observing the movement of the colored bands;
this is the origin of the term chromatography. If the components are colorless, the
progress of the separation may be followed by running TLC's on various arbitrarily
collected fractions, or by using UV light if one or more of the components fluoresces
under UV light. Fractions containing the same component are pooled and evaporated to
recover the individual components.
The more polar the compound, the more strongly it is adsorbed and the more slowly it is
eluted. Also, the more polar the eluent, the greater is its eluting power. The strengths of
adsorption of various classes of compounds and the eluting power of some common
solvents are given below. Sometimes a single solvent can be used as the eluent, but more
commonly the solvent polarity is systematically increased by changing the composition
of a mixture of two or more solvents.
RCO 2H
ROH
increasing
strength
of
adsorption
RNH2
O
RCR
RCO 2R
ROR
R 2C CR 2
RX (x = Cl, Br, I)
increasing
power
of
elution
methanol
ethanol
acetone
ethyl acetate
diethyl ether
chloroform
dichloromethane
toluene
hexane
petroleum ether
In this experiment a mixture of fluorene and fluorenone will be separated by column
chromatography using alumina as the adsorbent. One of the compounds is yellow,
whereas the other one is white. Thus the progress of the chromatography may be
followed to some extent visually by watching the movement of the yellow band.
O
Fluorene
Fluorenone
As a piece of chemical trivia, this hydrocarbon was named fluorene because of its
fluorescence when exposed to ultraviolet light.
Experimental Procedures
Preparation of the Column:
Vertically clamp a dry 25 mL buret that has a Teflon stopcock. Make sure that the
stopcock is closed. Push a small plug of polypropylene wool into the bottom of the buret
with a wooden dowel. Add enough clean, dry sand to form about a 1 cm layer on top of
the polypropylene wool, and then add enough petroleum ether (TBP = 30-60 oC) to halffill the buret. Open the stopcock and drain out a few milliliters of the solvent to remove
air from the sand and the polypropylene wool. Place a funnel on the top of the buret and
very slowly add approximately 6 g of dry alumina (no need to weigh: 1 teaspoon
measure, slightly heaping ≈ 6 g). When all of the alumina has been added, rinse the
inside of the buret with additional petroleum ether to flush down any alumina that may be
adhering to the walls. Add more clean, dry sand to form about a 1 cm layer on top of the
alumina. Carefully drain the solvent from the column until the solvent level just reaches
the top of the sand. The column is now ready for the addition of the mixture to be
separated.
solvent
sand
alumina
polypropylene
wool
sand
Separation of the Fluorene-Fluorenone Mixture:
Accurately weigh 0.2 g (~1/16 teaspoon) of the fluorene-fluorenone mixture, place it in a
small test tube, and dissolve it in 3 mL of petroleum ether. It will be necessary to slightly
warm the tube to effect dissolution. If upon warming the mixture has still not completely
dissolved, add three or four drops of dichloromethane, then re-heat. Repeat this step until
the mixture dissolves, being careful not to add too much dichloromethane.
Adding too much dichloromethane to dissolve the mixture will hasten the movement
of the yellow compound through the column and result in poor separation!
Carefully transfer this solution to the top of the sand layer inside the column with a
Pasteur pipet.
Transfer the solution while it is hot otherwise cooling may result in separation of
solid from solution!
Open the stopcock and slowly drain the column until the liquid level is at the top of the
layer of sand. From this point on, never allow the liquid level to drain below the top level
of the sand, or air voids and channels may develop in the alumina and ruin the
effectiveness of the column. Add another 2 mL of petroleum ether to the test tube,
transfer it to the column, and again drain the liquid level to the top of the sand layer. Fill
the buret with more petroleum ether, open the stopcock and collect the effluent (eluate) in
a 50 ml Erlenmeyer flask. Check the progress of the chromatography by collecting a
drop of the eluate on the bottom of a large upside-down beaker after every 5 mL of eluate
is collected. The solvent will rapidly evaporate and leave behind a solid residue if any
material is eluting from the column. This technique will allow you to determine when the
first component has been completely eluted. The majority of the first component should
elute in 15-20 mL of petroleum ether. Wash any residue that has collected on the tip of
the buret into the receiver with a small amount of petroleum ether.
After all of the first component has been eluted from the column, switch collection to a
second 50 mL Erlenmeyer flask. Continue eluting with petroleum ether until another 10
mL of eluate has been collected. Now change to dichloromethane as the eluent and
continue collecting the eluate in the second flask until the yellow band just reaches the
bottom of the alumina. Switch the collection flask to a third 50 mL Erlenmeyer flask and
continue eluting the column with dichloromethane until the eluate is colorless
(approximately 10-15 mL will be required).
The first and third fractions should contain pure components (either fluorene or
fluorenone). The second fraction should not contain any significant amounts of solid
material, but any present may consist of a mixture. Spot a TLC plate with all three
fractions and develop it using a 50:50 (V/V) mixture of petroleum ether/ dichloromethane
(already prepared and in TLC chambers). Remove the solvents from the first and last
fractions using either a steam plate in a fume hood or a rotary evaporator. If a rotary
evaporator is used, be sure to clean the glass adapter with acetone after each fraction is
evaporated to avoid cross-contamination. If a steam plate is used, remember to use a
boiling stick to avoid bumping. Determine the weights of the solid residues from these
two fractions. Assuming that the second fraction does not contain a significant amount of
residue, calculate a percent recovery (based upon the amount of the initial mixture used).
NEXT LAB: Determine the melting points of the pure components. Also determine a
melting point for the initial mixture and compare it to those of the pure components. You
will not be marked down for not reporting the melting points in the experimental section
(due day of column preparation and separation). Postlab information/questions
pertaining to the melting points of compounds (shaded areas) should be completed and
turned in with the summary one week after turning in the experimental section.
Points to note:
Spot each fraction (3 spots total). The middle fraction may need to be spotted several
times.
Use the glass disposal box for all glassware disposals except for mercury-containing
thermometers!
To save time, know the melting points of the pure products and determine all three
melting points simultaneously.
Name:______________________________ Section_______Date:_________
POSTLAB EXERCISE
COLUMN CHROMATOGRAPHY: SEPARATION OF A MIXTURE
OF FLUORENE AND FLUORENONE (8/3/05)
>> Due no later than 12 noon on the 2nd Friday following the scheduled experiment;
no exceptions will be made! Please answer questions on this form. (30pts TOTAL)
<<
Shaded areas should be completed (in lab) one week after completion of the
experimental portion of the notebook!
A. PRODUCT INFORMATION (15pts)
MP(oC) (6pts)
SAMPLE
Initial Mixture
(0pts)
Fraction 1
(3pts)
Fraction 3
(3pts)
% RECOVERY
X
(9pts) Indicate the appearance of your TLC plates and attach originals to this form. Be
sure to identify as many of the components as possible!
B. SUMMARY QUESTIONS (15pts)
1. (2pts) Based on the amount recovered from fractions 1 and 3, what was the
composition of the original mixture?
2. (2pts) Which of the two compounds separated in this experiment was yellow in
color?
3. (2pts) Was the melting point of the initial mixture higher than either of two pure
compounds? Why or why not?
(You may complete this question after completing the melting points.)
4. (3pts) Does the observed order of elution of fluorene and fluorenone agree with
the expected order of elution? Why?
5. (3pts) Why is it preferable to use a Teflon stopcock or an ungreased stopcock
instead of a greased stopcock when performing column chromatography?
6. (3pts) When a mixture is to be separated by column chromatography, why is it
better to switch from a less-polar to a more-polar eluent rather than the reverse
order?