Pre-lab preparation

Follow the routine procedure for preparing your notebook. Read through the procedure for both weeks and make sure all substances that you plan to work with (or produce) are listed in the required tables including the solvents used during the chromatographic separation and silica gel.

Hazard. Silica gel is a fine powder that, inhaled over time, causes a disease called "white lung" or silicosis. All operations with this powder should be performed in a fume hood. Also, the powder should be disposed of in a special container (located in same hood where it is dispensed).

Wear gloves and goggles and carry out all operations in a fume hood.

Reaction/Work up - Week 1

Combine ferrocene (4 mmol) and acetic anhydride (10-12 molar equivalents) in a test tube equipped with a stir bar. Add 0.5 mL 85% phosphoric acid to the mixture dropwise while attempting to stir the mixture. Note any changes.

Cool mixture to room temperature, then heat it in a boiling water bath for about 2-5 minutes [NOTE 1]. Note any changes.

Stop the reaction by pouring the mixture over 10 g ice [NOTE 2]. Stir this mixture until ice has completely melted. Neutralize the mixture by adding solid NaHCO₃ (about 5 g), then cool the mixture in an ice water bath for about 10 minutes [NOTE 3].

Vacuum filter the mixture using an appropriate sized funnel to collect the precipitate and rinse the solids with 3 x 15 mL of ice-cold water. Then draw air through the funnel to help dry the precipitate (10-15 minutes). Transfer the precipitate to a dry piece of filter paper and store it carefully in your lab cabinet while you prepare the dry-column (next section). Weigh the precipitate before you perform the chromatographic purification.

Dry column flash chromatography - Week 1&2

This separation requires several steps: passing material through a column and collecting fractions, identifying fractions that contain the compound(s) of interest, combining these fractions as seems appropriate, evaporating solvent from these fractions, and weighing the product(s).

We hope that you can get through all of these steps in one lab session. If this proves impossible, you must at least complete your work with the chromatographic column because columns do not store well, i.e., you must pass your material through the column, identify interesting fractions, and combine them.

Non-standard Version (NonStd). Acetylferrocene and ferrocene are easily separated using dry-column chromatography so we are going to cut a few corners in our procedure to save time. However, you will be using the dry-column method again next semester and it will necessary to perform the procedure more carefully at that time. Since you will probably refer back to these instructions when you next use this method, all shortcuts used in this experiment have been identified by the notation NonStd#. Following this link will lead you to a note that describes the current shortcut and tells you how to perform the procedure properly in general.

Preparing your column. Loosely fill the funnel to within 3-5 mm of the lip [NonStd1] with dry TLC-grade silica. Caution - silica powder is harmful if inhaled. Work carefully with this material and use it only in a fume hood.

Place your funnel on top of your filter flask (see below) and tap the funnel gently to settle the powder and to remove any voids. Next, apply suction and continue tapping the funnel gently on its side to remove voids, and then press down on the silica carefully, but firmly, with a flat stopper or rubber cork. Carefully work around the perimeter first and then in towards the center to produce a level, well-compacted bed. The goal is a reasonably flat, compacted surface with ~2 cm of head space [NonStd2] for the addition of the mixture and solvent fractions. When you are finished, release the vacuum.

Click on image for larger image

Place a small piece of filter paper (4 cm diameter) on top of the column to protect it from solvent (otherwise a crater will form each time you pour solvent on the column).

Test the packed column by rinsing it once with hexane. Do this by applying strong suction to the flask and adding hexane fast enough to keep the filter paper covered until the test is complete. As you do this, watch how the solvent "front" descends through the column [NOTE 4]. It should descend in a horizontal line, i.e., it should not percolate faster through one side than the other. Stop adding hexane when the entire column is wetted, but maintain suction until the solvent flow slows to a few drips.

Examine the "dry" column for channels, voids, holes, etc.. If any appear, discard the silica gel in the special waste container provided and repack the column with fresh silica gel and repeat the test. If the column passes the test, that is, if it is well-packed, move on to the next step.

Prepare and apply sample mixture. If you have not weighed your product, do so now [NonStd3]. Set aside a small amount of solid for TLC analysis later (5-10 mg suffices). Place the rest of your solids in a 100 mL beaker and dissolve them in 5-10 mL acetone [NonStd4]. Add 5 g TLC-grade silica gel to the solution and stir the resulting slurry until all of the acetone has evaporated. If necessary, you can accelerate the evaporation process by resting your beaker inside a larger beaker containing some warm water (be careful not to get any water in your silica gel slurry).

Remove the filter paper from your column. Place the dry silica gel-ferrocenes mixture on top of your dry column, replace the filter paper, and press down gently with a rubber cork or glass stopper to compact the added silica gel [NonStd5].

Elute the column. The general procedure for eluting the column involves the following sequence:

• Place a clean test tube in the filter flask
• Replace the funnel so that the drip tube extends partway into the test tube
• Apply strong suction (it may be necessary to adjust the funnel so that it makes a tight seal with the filter flask)
• Pour solvent (see below for solvent) evenly on top of the column until the test tube is partly full (about 25 mL, do not overfill)
• Carefully pry the funnel (or the rubber hose) from the filter flask to release the vacuum
• Remove and store the "filled" test tube

This procedure is repeated as many times as is necessary to elute ferrocene and acetylferrocene completely from the column. We cannot tell you exactly how many fractions will be required, so you should plan on collecting at least 16 fractions [NonStd6].

To separate your compounds, it is necessary to make a judicious selection of elution solvents. Ferrocene and acetylferrocene can be separated by applying the following solvents in sequence [NonStd7]:

• Fractions 1-4 - pure hexane
• Fractions 5-8 - 90:10 v/v hexane:ethyl acetate
• Fractions 9-12 - 80:20 v/v hexane:ethyl acetate
• Fractions 13-16 - 50:50 v/v hexane:ethyl acetate

Roughly 25 mL of solvent is required for each fraction, so it is convenient to mix up solvent for 4 fractions at once using a 100 mL graduated cylinder. For example, you can make up solvent for fractions 5-8 by adding 90 mL of hexane to a cylinder and then adding ethyl acetate. Make sure you mix the solvents well before applying them to a column.

Solvent and compounds in your mixture compete for binding sites on the silica gel. The gel is very polar so compounds that are more polar compete better and stay on the column longer. To put it another way, nonpolar compounds elute first, relatively nonpolar solvent mixtures elute nonpolar compounds, and more polar solvents are needed to elute more polar compounds.

It should be obvious when ferrocene and acetylferrocene elute because both compounds compounds are brightly colored [NonStd8]. However, it is necessary to analyze your "compound positive" (colored) fractions by TLC to see what they contain [NonStd8 - Round 2]. This is accomplished by eluting [NOTE 5] a sample of each positive (colored) fraction side-by-side with a sample of your original product mixture [NOTE 6].

Record the results of these TLC analyses in your notebook. Do not measure and record Rf values. Instead, make a drawing in your lab notebook that exactly duplicates your plate. Your drawing should accurately reproduce the size of your plate, the locations of the initial spots, the locations and sizes of the final spots, and the position of the solvent "front" at the end of the analysis. You should also include the developing solvent and label each lane. Padias Figure 3-29 shows what is expected (just leave off the distance measurements and calculated).

Ferrocene moves faster than acetylferrocene. Combine the fractions that contain only acetylferrocene in a round-bottom flask and evaporate the solvent from them using a rotary evaporator (Padias p. 144-145).

Weigh your dry purified product.

Use 10-20 mg to make an FT-NMR sample. Label it and leave it with your instructor.

Characterization - Week 2

Under construction (still need instructions for: NMR analysis & mp measurement)