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Lab III: Column Chromatography


Pre-lab Work

Reading Assignment:

  1. Column Chromatography
    Mohrig, Technique 17.1-17.6, pp. 206-219 & Appendix
    .
  2. Record the boiling points of the pure solvents used in this lab.
    [Reference Mohrig, Technique 4, pp. 34-38.]


Experimental Work Objectives:

  • To resolve a mixture of unknown composition in reasonable quantity
  • To identify the components of the mixture by their melting points and comparison to a list of possible unknowns.

You will be given a mixture of two of the 4 compounds listed in Table 2. This mixture can be separated using Column Chromatography. The separation will be monitored by Thin Layer Chromatography. Once separated, the melting points of the unknowns can be determined and the compounds can be identified using the information in Table 2.

Table 2: Melting Points for Possible Unknowns in Column Chromatography Lab

Compound Melting Point
Acetone 2,4-dinitrophenylhydrazone 124-126oC
Cyclopentanone 2,4-dinitrophenylhydrazone 144-146oC
4-Hydroxy-azobenzene 152-154oC
4-Methyl-2-pentanone 2,4-dinitrophenylhydrazone 79-81oC

Experimental Procedure
Separation and Identification of Binary Unknowns using Column Chromatography
Read this experiment all the way through and gather your material before you begin to work.

Eluting Solvents
The first eluting solvent you will need will be a 30% (by volume) solution of Ethyl Acetate in Petroleum Ether. Create 100-mL of this eluting solvent. Mix well. The second eluting solvent you will need will be a well-mixed 10% (by volume) solution of Methanol in Ethyl Acetate. Make approximately 50 mL of this solvent. Mix well. Store in covered containers to prevent absorption of moisture and/or evaporation.

Thin-layer chromatography (TLC) developing chamber and plate
Create a TLC developing chamber by placing a half-circle of filter paper and about 50 mL eluting solvent (30%
Ethyl Acetate in Petroleum Ether) in a 1500 mL beaker (your 'developing chamber.') Cover the 'chamber' with aluminum foil or plastic wrap. Using a pencil, label your alumina-coated TLC plate so that it has three lanes, one for your crude product and one for each of the two elutions you will obtain from your column. Store the plate in a clean place until needed.

Preparation of Binary Unknown for Column
Add approximately 0.5 mL of anhydrous ethyl acetate to your binary unknown mixture. You may need to heat over a steam bath for complete dissolution of the unknown mixture.

Preparation of the Column
Label three 50-mL beakers: Waste, Elution #1, and Elution #2. Using a long stirring rod, place a small plug of glass wool in the bottom of the column. Close the bottom of the column with a small piece of rubber tubing and a screw clamp. Fill the column 3/4 full with your eluting solvent. Slowly pour enough sand into the column to create an approximately 1-cm layer of sand on top of the glass wool. Pour approximately 10 grams of Alumina into the column on top of the sand. Lastly, create another approximately 1-cm layer of sand by pouring the sand slowly on top of the Alumina. When you are ready to add your mixture to the column, place your "Waste" beaker under the column and remove the tubing and clamp from the tip of the column allowing the solvent to flow freely for the rest of the experiment.

Addition of Mixture to Column
Let the eluting solvent drain to approximately 1 cm above the top of the sand. Using a long pipet, transfer the binary unknown/ethyl acetate mixture to the top of the sand layer making sure not to squirt your mixture onto the inner sides of the column. *Allow the mixture to adsorb into the top of the Alumina before adding more 30%
Ethyl Acetate in Petroleum Ether eluting solvent. Add small amounts of eluting solvent until the solvent reservoir above the top of the sand layer is colorless. Why?

Once you start adding solvent to the column, try to never let the solvent go below the top of the sand layer. Why? Once the small solvent reservoir is colorless, fill the column with solvent to help prevent the column from going dry.

*Save the residue in the vial for TLC analysis.

Eluting Unknowns from the Column
Continue to add 30%
Ethyl Acetate in Petroleum Ether to elute the first unknown into a labeled 50 mL beaker. After all of the first unknown has been removed from the column, switch to the second eluting solvent, 10% Methanol in Ethyl Acetate, and collect the second unknown into another labeled 50 mL beaker. Do not collect more than 40 mL of the 2nd elution. Finally, allow the column to drain into a waste beaker.

TLC
Add 0.5 mL ethyl acetate to the residue in your vial. Spot your prepared TLC plate with this solution of your unknown mixture and with Elution 1 and Elution 2. If the spots are not visible, check that you have placed enough of each compound on the plate by viewing the plate under a UV lamp. Develop the plate in your developing chamber. Immediately mark the solvent front when you remove your plate from the developing chamber. Examine your plate and comment on the purity of each elution. Use the UV lamp, if necessary. Calculate the Rf values for each compound.

Isolation and Identification of Solid Unknowns
Using a steam bath, boil off the solvent from both elutions. Remove the beakers from the steam bath immediately after the solvent has been evaporated. (How will you know when all of the solvent has evaporated?)

Identify the solids by taking their melting points and comparing them to the melting points in Table 2.

Special Waste Disposal
paper lined tote for used columns


Post-lab Work

  1. Identify the two components of your binary unknown mixture using melting points and the list of possible unknowns in Table 1.
  2. Knowing how the two compounds in your binary unknown mixture separated on the column, what do you know about how they differ chemically?
  3. What information did you get from TLC about the purity of your compounds?
  4. What information did you get from melting point about the purity of your compounds?
  5. Discuss any errors in your experiment and what you would do differently if you were to do this lab again.