Winthrop University Organic Chemistry LabDepartment of Chemistry CHEM 304Column ChromatographyColumn chromatography is an extremely important technique in organicchemistry. It is a versatile method used to separate and purify organic compounds. Andcan be used to purify both solids and liquids. Column chromatography is similar inprinciple to thin layer chromatography; compounds are separated based on their attractionto a solvent (mobile phase) and an adsorbant (stationary phase). Less polar compoundsare less attracted to the adsorbant and elute (travel) more quickly than polar compounds.In this experiment, you will assemble a column from a Pasteur pipet and use it to separatethe two components of a solid mixture of fluorene and fluorenone.Required Reading:Padías:Chromatography, general (pp. 150 – 152)Column Chromatography (pp. 160 – 165)Special Safety Notes:Alumina can be harmful to the lungs. Be careful to avoid inhalationProcedure:Place 25 mg of the fluorene/fluorenone mixture in a conical vial. Add a spin vaneand 0.3 mL of 5% dichloromethane in hexane. Stir to dissolve the solids. While themixture is stirring, obtain 9 mL of hexane, 2 mL of 30% acetone in hexane and 2 mL ofacetone in three separate 25 mL Erlenmeyer flasks. Place four test tubes in a rack andbegin preparing the column.Preparing the column:Place a very small plug of cotton into a 5.75" Pasteur pipet. Gently lodge thecotton plug into the narrowing region of the pipet using a length of wire. The cottonshould be pressed enough to secure it without excessive compression. Add 1.25 g ofalumina to the pipet and gently tap it on the bench to compact the solid.Revised 11/08Winthrop University Organic Chemistry LabDepartment of Chemistry CHEM 304Clamp the pipet to a ringstand just above Tube #1. Using a pipet, add hexane, inportions, to the top of the column (about 3 mL). Allow the hexane to flow through thecolumn. You will see the alumina begin to look "wet". Make certain that there is solventabove the alumina at all times. Continue to add hexane and allow it to flow through thecolumn until the alumina looks homogeneous and free of dry patches or cracks.Separating the Mixture:When the solvent reaches the top of the alumina, add the fluorene/fluorenonesolution using a pipet. When the solution reaches the top of the alumina, add another 1mL of hexane dropwise. Begin draining (eluting) the column into Tube #2, addinghexane until the yellow band nears the bottom of the column (about 5 mL). When thehexane reaches the top of the column, switch the solvent to 30% acetone in hexane. Justbefore the yellow band reaches the bottom of the column, begin draining the eluent intoTube #3, adding 30% acetone in hexane until the yellow band elutes completely from thecolumn.TLC Analysis:Obtain a 5 x 5 cm TLC plate and label it as shown using a pencil. Using a 1 Lmicropipette, spot Tube #2 and Tube #3 at the origin on lanes labeled #2 and #3 of theTLC plate. Spot standard solutions of fluorene and fluorenone on lanes labeled “ENE”and “ONE” of the TLC plate. Develop the plate in a covered beaker usinghexanes:dichloromethane (1:1) as the eluting solvent, removing the plate when thesolvent reaches the top line. Allow the eluent to evaporate from the plate and examine itunder UV light (254 nm). GENTLY circle each spot with a pencil. Draw the TLC plateto scale in your notebook, showing all spotsThings to Think About while Writing Your Report:For each plate calculate and tabulate Rf values for each spot (show yourcalculations). Identify each component and explain your reasoning.ReferencesPavia, D. L.; Lampman, G. M.; Kriz, G. S.; Engel, R. G. Introduction to OrganicLaboratory Techniques, A Microscale Approach; 3rd ed.; Brooks/Cole: Pacific Grove,CA, 1999; pp. 115 – 119.Revised 11/08#2 #3 ENE ONE0.5 cm1.0 cm0.5 cmWinthrop University Organic Chemistry LabDepartment of Chemistry CHEM 304Questions:1. If a column fraction contained two compounds (two spots upon TLC analysis),what could be done to separate the two compounds from one another?2. In this lab, why was the solvent changed in the middle of the columnchromatography procedure?3. 2-Naphthol can be treated with potassium hydroxide and the resulting productreacted with iodoethane to give a compound (Compound X) which is used as anorange blossom scent in perfumery.a) Give the structure of Compound Xb) If the reaction did not go to completion and you had to separate Compound Xfrom the starting 2-naphthol by column chromatography using a relativelynon-polar solvent, which compound would elute from the column first? Why?Revised