Day 3 - Thin-layer Chromatography of Acetylation Product
First determine the weight and take the melting point of the dried crude acetylation product.
Procedure for TLC
To determine the product distribution of the reaction mixture, an aliquot will be analyzed by thin-layer chromatography. TLC plates of aluminum oxide are available from the Stockroom. Applicators are micro-capillary tubes. Development is conveniently conducted in small jars (Fig. 3). A 5-1/2 cm diameter filter paper placed in the jar will saturate the chamber with solvent vapor. Be certain that the solvent level is below the point where the plate was initially spotted.
Prepare solutions of ferrocene (sublimed), the dried acetylation product, and a control solution containing both ferrocene and acetylation product by placing a few (5 to 10) drops of methylene chloride* CH2Cl2 in each of your three smallest sample vials. Add a small amount (one spatula-tip full) of ferrocene to one vial, the acetylation product to the second vial and a half spatula tip of both ferrocene and the acetylation product to the third vial (the control). The solution should be concentrated enough so that a dark spot is seen on the TLC plate when the sample is applied. Use the special micro capillary tubes to spot your plates. If you are unable to see the spots after developing, add more sample to the solutions.
Figure 3. Apparatus for the Separation of Acetylation Products using TLC
The goal of this portion of the experiment is to find the solvent system that most effectively separates the various ferrocene species. This is determined by examining the migration of reaction products in a variety of solvents of varying polarity. The following solvents will be used to analyze the ferrocene products: (1) hexane* and (2) ethyl acetate*.
Begin by preparing a chamber as shown in figure 3 for each of the following three solvent combinations 4:1, 1:1, and 1:4 ratios hexane:ethyl acetate. Students only need around 3mL of solvent (mobile phase) for each chamber. Plates should be developed until the solvent front is within 1 cm of the top of the plate. It should then be removed and the solvent line marked. If the progress of the solvent front is monitored closely, it is also acceptable to draw start and stop lines on the plate before the chromatogram is run (A pencil should be used because ink is soluble). This prevents inaccuracies that occur when the solvent evaporates before the final solvent front can be marked. Sketch the chromatograms in your lab notebook. Calculate Rf (“ratio to front”) values for each spot on the chromatograms. (Rf = distance a component travels/distance the solvent front travels.)
It is possible to optimize separation of reaction products by varying the polarity of the solvent systems. This is accomplished by adding hexane (non-polar) to ethyl acetate (polar). Make solutions of 4:1, 1:1, and 1:4 ratios of hexane: ethyl acetate. (If a greasy brown spot remains at the starting point no matter which solvent is used, be aware that this is an impurity and not diacetylferrocene.) Carry out TLC as above using these solvent mixtures.
By selecting a solvent that carries all products but still affords a clear separation, TLC can be used to quickly determine the qualitative makeup of a sample. However, when attempting column chromatography with a short column, such as one made from a Pasteur pipet, solvents that only carry one component at a time are preferred. Why? Include an explanation in your discussion. Save the remaining ferrocene and acetylation product/CH2Cl2 solutions for comparison by TLC with the purified products.
Based on your TLC results select a solvent system for use in the larger scale separation provided by column chromatography. Review your selection with your TA.
The semi-microscale column (MICROFLEX®, Williamson Microscale Kit), shown in figure 4, is used as a chromatographic column for separation of 20-200 mg mixtures. To separate the acetylferrocene from ferrocene (and 1,1-diacetylferrocene if present), prepare a column of acid washed alumina. * Clamp the empty column in a vertical position. Note: Because this column has a built-in frit, unlike standard-scale columns, it is not necessary to tamp down a plug of glass wool and add sand on top of it, before adding alumina.
Figure 4. Packing of a column chromatography
Add about 7-8 cm of the acid washed alumina (which has been previously dried in a 100 °C oven for at least 24 hours). Tap the column gently to pack down the alumina. Add 5 mm sand on the top of the alumina.
Apply the crude acetylation product to the column using the “dry-loading”. Dissolve the entire dry acetylation product in a minimum of methylene chloride (just a few drops) in an Erlenmeyer flask. Add about 50 mg of dry alumina and stir. In the hood evaporate the solvent completely until a fine, smooth flowing powder remains. Add this powder to the top of the column. Apply a 3 mm layer of sand to the top of the column. Add the solvent system chosen by TLC cautiously to avoid disturbing the sand; allowing it to pickup any particles adhered to the wall of the column (Note: The upper fitting serves as solvent reservoir). During the addition of solvent to your column keep the stopcock open to allow air to escape. Collect the eluant containing the first colored component in a weighed 10 mL Erlenmeyer flask. Change flasks and collect the second colored component in a second weighed 10-mL Erlenmeyer flask. (Note: the solvent coming off the column, which does not contain colored product, may be collected separately from the colored products). Be careful not to permit the column to go dry during this process. Expect to remove the same number of fractions, as the number of colored spots observed moving on the TLC plate. (Note: With this synthesis diacetylferrocene, if present at all, will likely be present in such small amounts that it cannot be recovered from the column).
Check the colored fractions by thin-layer chromatography to verify that each contains a single component. Evaporate the solvents in the hood using a very gentle airflow. Dry the crystals in your desk until Day 4 of the next lab session.