Lab I

Lab II

Lab III

Lab IV

Lab V

Lab VI

Lab VII

Lab VIII

Lab IX

Lab X

Lab XI

Lab XII

Lab II: Column Chromatography

Pre-lab Work

Reading Assignment:

1. Column Chromatography
   Mohrig, Technique 17.1-17.6, pp. 206-219 or Technique 18.1-18.6 pp. 236-248 and Appendix.
2. Record the boiling points of the pure solvents used in this lab.
   [Reference Mohrig, 2nd edition, Technique 4, pp. 34-38 or 3rd edition Technique 3.3 pp. 25-28 to find paper and online sources for obtaining physical property information (i.e.-boiling points) about organic compounds.]

Pre-lab Questions:
(Please turn in the answers to these questions when you arrive to lab.)

1. Please describe the procedure you will use to create 50-mL of a 10% (by volume) methanol solution in ethyl acetate.  (Note: since the solution concentration has only two sig. figs in it (10%), the precision with which you make this solution does not have to be very precise.)

2. What effect would a slight impurity (<10%) have on the melting point of a solid and why? (Note…check out the concept of “Freezing Point Depression” from intro. chem. stuff.)

3. What are the boiling points of all the individual solvents you will be using in this lab and list them in order of least polar to most polar?  (Dielectric constants are a measure of a compound’s relative polarity…check Mohrig, Table 9.1.)

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

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 30 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. What's the purpose of the filter paper? 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. What's the purpose of this plug of glass wool? 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. What's the purpose of this bottom layer of sand? 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. What's the purpose of this top layer of sand? 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. Is it OK to mix the two eluting solvents when switching from one eluting solvent to the other eluting solvent? Why or why not?

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
Follow the instructions of your lab instructor to evaporate the solvent from your two elutions.

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

Waste Disposal and Clean-up

1. Place used columns in the paper-lined tote in the hood.
   
2. Rinse your glassware with acetone and dispose of the acetone rinse in the flammables waste bottle. Place developing beakers back in their original tote and place all other beakers in the dishwashing tote.

Post-lab Questions
(Please turn in the answers to these questions when you arrive at your NEXT lab.)

1. In a data table, please report your unknown number and what the two components of your binary unkown mixture were from the list of possible unknowns in Table 2. Please justify your answer. [Please report the voltage/power level that was used to obtain your melting points and any observations you took while recording the melting points.]
   
2. In your TLC analysis, how many spots did you see under UV for your crude unknown mixture? How many spots did you see for each elution? What does this tell you about the purity of your eluted compounds?
3. Knowing how the two compounds in your binary unknown mixture separated on the column, what do you know about how they differ chemically?
   
4. Based on what you know about freezing point depression and wide mp ranges due to small amounts of impurities, do the melting points for each of your unknowns support or contradict your TLC results? Explain.
5. How would you separate and isolate two colorless compounds using only column chromatography and TLC?
6. Discuss any unexpected results in your experiment. What you would do differently to improve your results if you were to do this lab again.