Column Chromatography

Purpose: Separate a mixture of fluorene and fluorene by column chromatography

Read: Mayo, Technique 6 Chromatography including the following sections: Column Chromatography, Packing the Column, Sample Application, Elution of the Column, and Fraction Collection. Look up and record the structures and melting points of fluorene and fluorenone. 

Background: Like thin layer chromatography, silica gel is used as a stationary phase and an organic solvent, less polar than the silica gel, is used as the mobile phase. Column chromatography is carried out in a glass tube clamped vertically with the initial mixture placed at the top. Organic solvents run past this mixture on their way down the column. Because of silica gel's stronger affinity for the more polar components, the components with the lower polarity will descend ("elute") first through the column. During the chromatography, the polarity of the solvent's polar character is slowly increased by varying the solvent mixture used. As a result, increasingly polar components will elute.  
    As in any other chromatography it is important to introduce the mixture in as concentrated volume as possible to avoid overlapping of the components as they elute. 
    Below the column, preweighed flasks are used to collect the solvent with the various components ("fractions"). Normally flasks are changed when the solvent mixture is changed. Each flask is then warmed to evaporate the solvent, then reweighed, and the amount of component in the fraction is recorded. 
    In the procedure below a two component mixture will be separated. Ideally the two components will end up in separate flasks, preferably with an empty flask in between. This blank fraction is akin to having a baseline separation of peaks on a gas chromatograph chart or a clear space between spots on a TLC.
    Success in a separation requires good organization and planning, a concentrated mixture at the beginning, and as few interruptions of dripping from the column as possible after the chromatography is started. 

Procedure: The class will divide itself into teams of 2 (consult instructor) to carry out a column chromatography. Divide the work to reduce time spent; steps 1 and 2 can be carried out simultaneously:

1) Obtain six 50 mL Erlenmeyer flasks and/or beakers; use a brush to wash each with soapy water. Thoroughly rinse each flask with distilled water then a small amount of acetone and then ether; shake out the ether and then warm each briefly on the steam bath and tip them to allow the ether vapors to escape. The flasks should be clean and dry. Weigh each to the nearest milligram and mark this with a felt pen on each flask.

2) Obtain a clean 5 mm x 20 cm column with a fritted disk (if you do not have a fritted disk, then you must put in some glass wool, else your silica will slip out),  and fill it with dry silica gel. Pour the silica gel back out into a 100 mL beaker and add 20 mL of petroleum ether to the beaker (Note: petroleum "ether" is really a mixture of alkanes whose boiling point is 30-60 oC. Do not confuse this with diethyl ether, C2H5OC2H5, that is normally used as a solvent) Stir the contents of the beaker to remove bubbles, check to make sure that you have a fritted disk, or some glass wool at the bottom of your column, then transfer the silica slurry back into the column. Avoid pouring the silica gel by itself; this will cause the stationary phase in the column to slow the flow of the mobile phase. If necessary, recycle the petroleum ether that drains to transfer the silica gel to form a 18-20 cm column. Pour  10 mL of recycled petroleum ether then 10 mL of fresh petroleum ether down the sides of the tube so as not to cut channels in the silica gel stationary phase.  Be sure all inside surfaces are rinsed and drain and discard the solvent. The column's flow can be paused briefly but it is wise to begin the next step right away. Do not let your column ever dry out during the separation process.

3) Weigh approximately 500 mg of the fluorene/fluorenone mixture to the nearest milligram into a 25 mL Erlenmeyer. Add 3 drops of chloroform to dissolve as much of the mixture as possible. Carefully transfer the solution directly to the top of the silica gel in the column (not down the side of the glass tube). Add another 3 drops and repeat the process. Repeat once more if necessary, but excess chloroform in the column at this time will make the eluting solvent too polar. Prepare 10 ml of 9:1 (v:v) pet ether:chloroform and pass this solution through the column. Collect the eluate (the liquid that has passed out the stopcock) with one of the clean, pre-weighed Erlenmeyer flasks. As the final milliliters of 9:1 petroleum ether:chloroform descend, prepare and add 10 ml of 4:1 pet ether:chloroform (fraction 2). For each fraction, remove the solvent by evaporation as described above (steam bath, splinter, remove splinter, remove solvent fumes) and re-weigh. The results are tabulated with headings (left to right): Fraction Number, Solvent, Mass, Remarks (such as "crystals -light yellow, mp of.." or "oil" or "nothing" or "not enough to analyze"). Fraction 3 is eluted with 1:1 petroleum ether:chloroform, and fraction 4 and 5 are eluted with pure chloroform.  A final fraction with reagent (not washing grade) acetone may be necessary to strip the final fraction (yellow - an extra pre-weighed Erlenmeyer flask is prepared as a back-up.)

Thin Layer Chromatography (TLC):  Each group has to perform two TLC plates.

1.  This can be done while waiting for column chromatography fractions (step 3) below. Place and develop three spots on a silica gel TLC plate: pure fluorene, pure fluorenone, (from the reagent shelf) and the mixture you are to separate. Use a developing jar with a 9:1 mixture of hexane:chloroform as the developing solvent.  Repeat until a good TLC plate is obtained.

2.  For the second plate, spot all the fractions that you collected above and the mixture of fluorene and fluorenone.  From the TLC plate, indicate which fractions are pure in your results and discussion. 

Repeat the chromatography as necessary to obtain a clean separation of each of the components.  You should provide a satisfactory accounting of the amounts submitted and obtained from the chromatographic process.

Each group should obtain and melting points and interpret IR spectra of the two compounds found among the fractions obtained. Pay particular attention to absorptions at 1715 cm-1 for one compound and at 2950 cm-1 for the other. Using available spectra from the Internet, check your samples for contamination from the other compound in the mixture you separated.

Some points to be addressed in your report:

1. From the TLC of the starting mixture, what are the approximate amounts of fluorene and of fluorenone? 

2. Were the relative Rf values as you expected? Explain.

3. From the melting points of the fractions obtained in the column chromatography, was the order of elution (i.e., fluorenone before fluorene or vice versa) in the order you expected? Why?

4.  Using the masses of the fractions from the column chromatography, give a more quantitative figure for the percents of fluorene and fluorenone.

5. Did the masses of the fractions sum to the mass of mixture you submitted to column chromatography?

6.  Do your IR spectra support the notion as to which compound (the more polar or less polar) should elute first from the column?   Explain.

References:

"Mayo et al.": Mayo, D.W., Pike, R.M., Butcher, S.S. and Trumper, P.K. Microscale Techniques for the Organic Laboratory; Wiley: New York, 1991

"Handbook": any of the recent editions of: Weast, R.D. Handbook of Chemistry and Physics; The Chemical Rubber Co.: Cleveland, 1960-present.

"Aldrich Price List": any addition of Aldrich Handbook of Fine Chemicals; Aldrich Chemical Co.: Milwaukee, 1970-present.

Revised Spring, 2006