Notes on Procedure for Experiment No. 25 – Week 2 The first thing to do is to weigh the flask, stir bar, and caffeine from last week. Then use a spatula to place a few crystals of the caffeine between two microscopic glass covers. In the small classes, everyone will now determine the melting point of their caffeine. The melting point apparati will be on the back desk. Your TA will show you how to use them. They have a magnifier, and a light will shine onto the sample when you turn on the apparatus. There is also a piece of paper attached, which gives a rough estimate of the temperature that will be reached at each dial setting. You can heat up to 100 degrees quickly, and then proceed more slowly to the melting point. Your sample will probably be rather impure, so the melting will have a wide range between the temperature at which the sample just begins to melt and all of it has melted. Record the temperature range of melting. Turn off the apparatus and dispose of the sample in the waste beaker by the apparatus. BE CAREFUL NOT TO BURN YOURSELF, SINCE THE FINAL TEMPERATURE WILL BE CLOSE TO 200 DEGREES. There are only six apparaati. Therefore, in the larger classes, the melting determination will be done in two groups. While the first group is measuring the melting point, the second group will start the experimental part (preparing the column, using it, preparing the TLC plate, and developing it). The melting point apparatus cools slowly. A good time for the second group to do their melting point is while the TLC plates are drying after development. Each student will prepare a silica gel column, as shown in Figure 3.27a on page 62 of your lab manual. Use the tubes in the beaker under the window. They are similar to Pasteur pipets, but work better. Most will still have the glass wool plug from the previous use that you can re-use. It is important to use the amount of silica gel given. Weigh on a creased weighing paper and carefully pour it in above a second weighing paper so that if you spill any, you can recover it. There should be a small three-pronged clamp in your desk to use. Wet the column with methylene chloride. Then dissolve the caffeine in a small amount of methylene chloride and transfer about 5 to 10 drops to the column. Collect anything coming off the column as waste. The caffeine should now be on the column. When the liquid level reaches the top sand layer, add 0.5 mL of the 5% solution and catch what comes off as waste. When the level reaches the sand layer, the 5% solution now occupies the column. Next, add 0.5 mL of the 10% solution. It will push out the 5% solution, which you should catch in your 5%-marked test tube. Again, when the level gets to the sand, add 0.5 mL of the 20% solution and catch the eluted 10% solution. Continue until you have collected all five of the solutions. You will have to put on two portions of the 100% solution in order to get the first 100% portion off. There will be volumetric flasks with the ethyl acetate/methylene chloride solutions in the left-hand fume hood. There will also be three or four labeled 5 mL graduated cylinders (with pipets) for each solution. Work in groups of three to five students to share these solutions. Get 3 or 4 mL of each solution. When needed, use the pipet to transfer the 0.5 mL of solution to the column, and then pass the graduated cylinder of solution on to other members of the group. While you are waiting for each potion of solution to pass through the column, you can be preparing the TLC plates. Get two (about 2 by 4 inches) TLC’s from the desiccator. Handle them by their edges. The shiny side is the plastic backing, and the dull side is the silica gel coating. Figure E26.3 on page 526 of your lab manual gives you some idea of the final appearance, but it is better if you do not draw a pencil line across the bottom at 1 cm as it is too easy to scratch through the silica gel layer, which slows the development later. It is better to make a little tic mark at each side to guide you where to put the spots. If you make a mistake, rotate the TLC plate and use the other end. Use six separate capillary tubes to spot the solutions onto the TLC plate (the five solutions in the test tubes and a known solution of caffeine), three solutions per TLC. Your TA will demonstrate the technique. To develop the TLC’s, prop them in a 400 mL beaker, add the developing solvent, and cover with plastic wrap, using a rubber band to keep the wrap tight. Make certain that the solvent is below the spots. Try not to move the beaker while developing. When the solvent gets close to the top of the plates, take them out of the beaker, immediately mark the solvent front with a pencil and place on a paper towel under the hood to dry. If you are in the second group in a larger class, now would be a good time to measure the melting point of your caffeine. When the TLC’s have dried, take them into the front of the balance room. There will be a couple of UV lamps there. Examine the TLC’s under the UV light to find and mark the spots of caffeine. You are mainly interested in which solvent mixture(s) eluted (removed) the most caffeine from the column. DO NOT LOOK DIRECTLY AT THE UV LIGHT AS IT CAN DAMAGE YOUR EYES. Pour all solvents into the waste jug and put the used columns into the beaker in the hood. DO NOT RINSE THE 400 mL BEAKER IN THE SINK, but put it uncovered under the hood at your desk. Ask your TA if you should include the TLC’s with your report.
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