EXPERIMENT 2 USE OF ORGANIC TECHNIQUES TO SEPARATE A MIXTURE WEEK 2: COLUMN CHROMATOGRAPHY.
Part 1: Thin Layer Chromatography Part 2: Purification of an Unknown Mixture via Column Chromatography Part 3: Using Melting Point to Determine Purity
Reading Assignment: Pavia Sections 9.1 – 9.5, 9.7 – 9.8; 19.1 – 19.5, 19.6B, 19.8- 19.10, 20.1, 20.2, 20.4B, 20.5 – 20.7, 20.9, 20.10.
Pre-lab Questions: (due week 2 prior to your lab) 1) Pavia page 127, problem 4. 2) Give a brief definition of each of the following terms associated with TLC: a.) Rf value; b.) origin; c.) spotting; d.) development; e.) visualization. 2) Pavia page 326, problems 3, 4, 5.
Part 1: Thin Layer Chromatography
1) Set up three clean, dry test tubes and label them as crude, recrystallized, and extracted solid.
2) Transfer a small amount (the tip of a spatula) of crude sample into the first test tube. Transfer a spatula-tip full of the recrystallized sample into the second test tube, and transfer a spatula-tip full of the extracted sample into the third test tube.
3) Add 1 mL methodicalness to each tube, and mix well.
4) Prepare your TLC chamber by filling it with a solution of hexane:ethyl acetate (4:1) to a depth of 0.8 cm. This solution is your eluent. Easy way to do this: using a 10 mL graduated cylinder, add 8 mL hexane and 2 mL ethyl acetate, then thoroughly mix. This will give you more than you will need, but will allow you to replenish the eluent as it evaporates. The TLC chamber is a screw-cap jar.
5) Place a half piece of filter paper around and touching the inside of the chamber. Give the other half paper to another student. At the end of the period, keep your paper in the chamber for use in later experiments. The filter paper acts as a wick from which solvent evaporates to saturate the air space. The chamber should stand covered for several minutes before use to ensure this process is complete.
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6) One centimeter from the short edge of a small TLC plate, make a light pencil mark at left (no pen!). The mark defines the origin. Do not touch the adsorbent surface at any time during the experiment; oils from the skin may contaminate the plates.
7) Next to the mark, in the positions indicated in the drawing to the right (C for crude, R for recrystallized and E for extraction), spot the samples (one spot each). Keep the spot small by touching the TLC spotter gently to the silica and lifting immediately. If in spite of your care the spot is too large (as observed under UV light in step 8), you can try again. Allow the plate to dry between each of a series of spotting, and at the end before placing in the developing chamber.
8) Check the plate under short wavelength UV light. DO NOT LOOK DIRECTLY AT THE UV LAMP – YOUR EYES MAY “SUNBURN.” Some materials can be visualized under UV light. For these, you can tell if they are present where wanted (at the origin where you spotted), present where unwanted (contaminating the plate at other locations), or absent. You can of course get no information for UV-invisible materials by this method.
9) Using forceps, carefully place the TLC plate almost vertically in the development chamber on the side away from the filter paper. Make sure the TLC plate does not touch the filter paper. If you have measured as instructed, the spots will initially be above the level of the solvent. The plate should not touch any surface along its side; it rests against the glass at the top and bottom. As development progresses, the solvent will be drawn up the plate by capillary action.
10) When the solvent front has advanced to about 1/2 cm from the top edge, remove the plate from the development chamber. Because of quick evaporation, draw a pencil line along the solvent front immediately as you remove the plate from the chamber. Cap the developing chamber. After the TLC plate has dried, examine the plate by short wavelength UV light.
11) With a pencil, carefully outline the spots. Use a dotted outline for faint spots, if any are observed. In your notebook (not on the plate itself), record whether spots are fluorescent (bright against the background) or absorbent (dark). Are there any spots that are visible with the naked eye, but not visible under UV light? Take a picture of the TLC plate with your phone and upload into you ELN.
Before & After TLC Development pencil mark C R solvent front E C R E
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12) Using forceps, place the plate in the iodine chamber. Let it remain for about five minutes. IODINE SOLID AND ITS VAPOR ARE CORROSIVE. AVOID SKIN CONTACT AND INHALATION. KEEP THE IODINE CHAMBER IN THE HOOD, CLOSED AS MUCH AS POSSIBLE. Because the chamber should be saturated with vapor, the air space should appear purple – if not, report the problem to your instructor. In your ELN, record whether or not the spots were visualized by iodine. The information should be recorded within a few minutes of treatment, since the color gradually fades. Are there any differences between the three samples? Are all of the spots visible under UV light? What about after sitting in the iodine chamber? Can you see any of the spots without iodine or UV light? Are you able to determine purity using TLC? Again take a picture of the TLC plate with your phone and upload into you ELN.
13) Record the information you will need for Rf calculations:
Rf = distance traveled by compound distance traveled by solvent
A good way to do this is to take a picture of the TLC next to a ruler. Rf values may be calculated after lab and are not required to be recorded in your notebook.
14) Now repeat steps 6 – 13, this time spotting the TLC plate with recrystallized sample, and your supernatant (mother liquor) from last week. After the plate has developed, how many spots do you see for your recrystallized sample? How many spots for the mother liquor? Can you tell by TLC whether the recrystallized solid is more pure than the supernatant?
Part 2: Purification of the Crude sample using Column Chromatography
1) Weigh and transfer approximately 0.1 g of your crude sample into a small test tube. Dissolve in a minimum amount of acetone (do not exceed 2 mL acetone – due to acetone’s polarity, adding too much will can damage the column and the separation.)
2) Place 5 medium test tubes in a test tube tray, and label as 1, 2, 3, 4 and 5.
3) Slurry Pack the Column: Fill the chromatography column with about 3 inches silica gel, then pour the silica gel into a small beaker. DO NOT BREATHE IN SILICA GEL! All manipulations with silica gel must be done in the hood.
4) Clamp the column in the hood.
5) Prepare about 25 mL of an hexane:ethyl acetate (4:1) solution. Great way to do this: use a 50 mL graduated cylinder and mix enough for you and your hood-mate. Think: how much hexane and ethyl acetate do you need to make 50 mL of a 4:1 solution? Calculate this before you go to lab!
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6) Pour some of this solution into the beaker containing the silica gel, swirl rapidly, then quickly pour this silica gel/solvent slurry into the column. Rinse the beaker with several portions of solvent to get most of the silica gel into the column.
7) Place the beaker under the stopcock, then open the stopcock of the column and allow the eluent to run through. You can connect an air hose to the adapter on the top of the column and allow a gentle stream of air to help push the eluent through the column. When the solvent reaches the top of the silica gel, (DO NOT LET THE TOP OF THE SILICA GEL GET DRY-It must be wet with solvent at all times) close the stopcock.
8) Introduce the crude sample from step 1 to the top of the silica gel using a pipette. Be careful to introduce the sample carefully so that the top of the silica gel stays flat and level. Open the stopcock and allow the solvent to drain just until it reaches the top of the silica gel. Close the stopcock. Rinse with a few portions of eluent, then open the stopcock to allow the solvent to drain just until it reaches the top of the silica gel. Close the stopcock.
9) When the crude sample is completely adsorbed onto the silica gel, and there is no crude sample clinging to sides of the column, add eluent carefully until it almost fills the column. Place the test tube tray underneath the column so that the solvent will go into test tube #1, and open the stopcock.
10) Allow the eluent to run through the column (you can use a gentle stream of air to help push the eluent through the column.) Fill each test tube about ¾ full (this is known as “collecting fractions.”) Collect fractions until a colored band can be observed in the middle of a column (no more than 5 fractions), then close the stopcock.
11) On a large TLC plate (if available – otherwise use multiple small plates), spot the fractions alongside the crude sample and the previously recrystallized sample to determine if you have collected pure unknown compound, and in which fractions it is contained. Develop the TLC plate and visualize with UV light.
12) Combine the fractions that contain pure unknown into a pre-weighed round bottom flask, then remove the solvent by rotary evaporation.
13) Determine the weight and percent recovery of the purified unknown. Make note of the solid form (powder, crystals, etc.) and color.
Part 3: Melting Point
1) Follow the procedure below (step 2) for acquiring melting points and determine the melting points for:
1st run:
a. The biphenyl/naphthalene purified by recrystallization. b. The biphenyl/naphthalene purified by extraction c. The biphenyl purified by column chromatography
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2nd run:
a. The original sample (crude) (if crude sample doesn’t melt by 150°C, stop at 150°C.) b. A mixed melting point of the purest sample from the first run & naphthalene. c. A mixed melting point of the purest sample from the first run & biphenyl.
2) Procedure for melting point determination: Take a thin-walled capillary tube that is sealed at one end and fill it to a height of 1-2 mm with sample that has been ground into a fine power in a small mortar and pestle. For a mixed melting point (2nd run, b, c), mix equal amounts of your purified sample and either naphthalene or biphenyl in a small mortar and pestle, and pulverize until well-mixed and finely powdered.) A mixed melting point will enable you to determine whether you have naphthalene or biphenyl (see section 9.4 in Pavia.)
3) Start heating the melting point apparatus. You can heat rapidly to within 15°C of the expected melting point, but then you must slow down to about 1 – 2°C per minute. Heating faster than this will give an inaccurate melting point because the temperature within the capillary tube will not have time to reach thermal equilibrium. Heating too fast also will make the melting point apparatus really hot, and you will have to wait a long time for it to cool down to make another run.
4) Record the melting point for each of the samples, being sure to note the full melting range. How do the melting points of the purified samples compare with the crude sample? Based on the results of 2nd run b,c, do you have biphenyl or naphthalene?
Results and Discussion:
Results: Summarize all results in a neatly organized table. Show sample calculations.
Discussion: Compare and contrast the three different methods for purification of the crude unknown mixture. Which procedure gave the best recovery? Is this what you would expect? Which method gave the purest sample? How do you know which gave the purest sample? Which method was fastest? Discuss the advantages/limitations of each of the three methods. Briefly discuss whether you think the results are a reflection of your abilities or of the procedure itself?
Keep in mind that different techniques may be best for biphenyl vs. naphthalene.
Post-lab Questions: (due week 3 at the beginning of your lab) 1) Inexperienced workers commonly make mistakes in TLC experiments. Give the result in the following case: * You spotted 1/2 cm from the bottom edge and immersed the plate in 1 cm depth of solvent. 2) One of the most common causes of inaccurate melting points is heating of the melting-point apparatus too rapidly. Under these circumstances, how will the observed melting point compare with the true melting point?
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3) Pavia, page 326, problem 8. 4) Pavia, pages 311- 312, problems 3, 4.
