ࡱ> %` #bjbj"x"x 6@@!JJJJ$L...D  3555555$jhDY"YJJ.DnJ8.D33D 0330SF4YY2 d2 JJJJJJ TLC Introduction Chromatography is a sophisticated method of separating mixtures of two or more compounds. The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving. Chromatography works on the principle that different compounds will have different solubilities and adsorption to the two phases between which they are to be partitioned. Thin Layer Chromatography (TLC) is a solid-liquid technique in which the two phases are a solid (stationary phase) and a liquid (moving phase). Solids most commonly used in chromatography are silica gel (SiO2 x H2O) and alumina (Al2O3 x H2O). Both of these adsorbents are polar, but alumina is more so. Silica is also acidic. Alumina is available in neutral, basic, or acidic forms. Thin Layer Chromatography (TLC) is a sensitive, fast, simple and inexpensive analytical technique. It is a micro technique; as little as 10-9g of material can be detected, although the sample size is from 1 to 100x10-6 g. TLC involves spotting the sample to be analyzed near one end of a sheet of glass or plastic that is coated with a thin layer of an adsorbent. The sheet, which can be the size of a microscope slide, is placed on end in a covered jar containing a shallow layer of solvent. As the solvent rises by capillary action up through the adsorbent, differential partitioning occurs between the components of the mixture dissolved in the solvent the stationary adsorbent phase. The more strongly a given component of a mixture is adsorbed onto the stationary phase, the less time it will spend in the mobile phase and the more slowly it will migrate up the plate. The following are some common uses of Thin-Layer Chromatography: To determine the number of components in a mixture. To determine the identity of two substances. To monitor the progress of a reaction. To determine the effectiveness of a purification. To determine the appropriate conditions for a column chromatographic separation. To monitor column chromatography. In this experiment, you will use TLC to identify unknown analgesic painkillers using the table of analgesics and their components in the experimental section of this experiment. Apparatus  INCLUDEPICTURE "http://www.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/materials.jpg" \* MERGEFORMATINET  Experimental Procedure: TLC Analysis of Analgesic Drugs Obtain 2 TLC plates. Draw a light pencil line about 1 cm from the end of each chromatographic plate. Spot one plate with your 4 known standards (Acetaminophen, Aspirin, Caffeine, and Ibuprofen) and the other plate with the 5 unknown commercial painkillers. Both plates should also have a Reference spot that contains all 4 standards. Use a separate capillary tube for each standard and unknown solution. Make each spot as small as possible, preferably less than 0.5 mm in diameter. Examine the plate under the ultraviolet (UV) light to see that enough of each compound has been applied; if not, add more. The standards and commercial painkillers will be dissolved in a 50/50 Ethanol/Ethyl Acetate solution.  INCLUDEPICTURE "http://www.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/developing.jpg" \* MERGEFORMATINET  Prepare a developing chamber as indicated in the picture using a large beaker as the chamber, a half-piece of filter paper inside, and foil or plastic wrap to cover. Pour the eluting solvent, a 99/1 mixture of Ethyl Acetate/Glacial Acetic Acid, into the beaker to a depth of approximately 1 cm. Place the prepared TLC plates in the developing chamber. After the solvent has risen to near the top of the plate (about 1 cm from the top), remove the plate and mark the solvent front with a pencil. Keep the plates in the hood until the majority of the eluting solvent has evaporated from the plates. Examine the plate under UV light to see the components as dark spots against a bright green-blue background.  INCLUDEPICTURE "http://www.wellesley.edu/Chemistry/chem211lab/Orgo_Lab_Manual/Appendix/Techniques/TLC/developedplatesuv.png" \* MERGEFORMATINET  Outline the spots with a pencil and note anything distinctive about any of the compounds. The spots should also be visualized by putting the plate in an iodine chamber. The iodine chamber is pre-made and contains a few crystals of iodine in the bottom of a capped jar. More than 2 plates can be placed in the iodine chamber at one time. Remove the plates when a definite change in appearence takes place on your plates. Note which compounds stained with iodine and to what intensity. The iodine stains will dissipate over time. Wrap your TLC plates in plastic wrap and scan them into your e-lab. Calculate the Rf values for each spot. Unknowns can be identified using Rf values, fluorescence in UV light, changes due to iodine exposure, the reference spot MAKING TLC CHAMBER To prepare a developing chamber, obtain a piece of 11 cm filter paper, and fold it about 1 inch from the edge, to obtain a flat edge. Place the filter paper, flat edge down, in your 400 mL beaker. Obtain 15 mL of the TLC solvent, Ethanol/Ethyl Acetate solution, and pour it into the beaker, swirling the beaker to wet the filter paper with the solvent. Carefully place the TLC plate in the developing chamber, spotted side down, trying not to splash the solvent on the plate. The level of solvent must be below the pencil line. Cover the beaker with a watch glass. The solvent will rise up through the stationary phase on the plate. When the solvent has risen to 1-2 cm from the top of the plate, remove the plate, and draw a light pencil line across the plate, at the level to which the solvent rose. Allow the solvent to evaporate (waving the plate in the air will speed this up), then look at the plate under the UV light. Circle all of the spots. TLC Plate TLC Developing Chamber  EMBED ACD.ChemSketch.20   You will identify the components of your unknown tablet by comparing the amounts the components traveled up the plate with the amounts the standards traveled. These amounts are reported as Rf (retention factor) values. X Distance the spot traveled Rf = --- = ------------------------------------ Y Distance the solvent traveled Measure the distance a spot moves from the center of the spot. A developed plate may look like this: From the locations of the spots, it would appear that the Your Aspirin (YA) contains Aspirin (A) and acetanilide (Ac). Perhaps you didnt do a good job of separation in the extraction. Your unknown (YU) only contains Ac. The spot for Ac in the standard looks larger than the spot for Ac in YU: this is probably due to different amounts of material being spotted on the plate initially. The Rf value for the A spot would be X/Y: measure X from the middle of the spot. Separate Rf values are calculated for each of the spots in YA. Since the spots for the materials in the samples and in the standards are different sizes and shapes, they may have slightly different Rf values.  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