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Experiments for 7 Experiment 1: THIN LAYER CHROMATOGRAPHY: INTRODUCTION: Chromatography is defined as a method for the separation of a mixture into its components by distribution between a mobile phase and a stationary phase. There are many variants of this technique used in organic chemistry, but our focus will be on Thin Layer Chromatography (TLC). In TLC the stationary phase is a finely divided solid (typically silica gel or alumina), and the mobile phase is a liquid. The mixture to be separated is applied to the stationary phase at the bottom end of the TLC plate. The mobile phase solvent is allowed to move up TLC plate by capillarity, carrying the components of the mixture with it. The rates at which the various components move along the path through the stationary phase will vary, depending upon their solubility in the mobile phase, and the strength with which molecules of the different substances are adsorbed on the surface of the stationary phase particles. In TLC the stationary phase material is coated as a thin layer on an inert support such as a glass plate or sheet of plastic or aluminum. A sheet or strip of the support material which is coated in this way is called the "thin-layer chromatographic plate" or, simply, the TLC plate. TLC is most commonly used as a very sensitive analytical technique, capable of analyzing samples at the microgram level. However, if plates of larger size are used, and if the coating of stationary phase material is thicker, it may be possible to separate quantities on a small preparative scale, up to 100 mg or more. The discussions below are intended to be rather general in nature, and specific instructions will be included with experiments where chromatographic separations are applied. DISCUSSION: TLC plates may be prepared in the laboratory by a variety of techniques. These usually involve the preparation of a slurry of the stationary phase material, such as finely divided silica gel (SiO2) or alumina (Al2O3), in an appropriate solvent. The slurry is then coated in a uniform layer on the surface of a clean glass plate. When the solvent has evaporated the silica gel or alumina remains as a layer on the surface of the plate. "Binders" such as plaster of Paris are sometimes incorporated with the silica gel or alumina to enhance the durability of the layer. Substances that serve as "indicators" may be included to make it easier to locate the positions of components on the plate after it has been developed. Pre-coated TLC plates, which we will use in this experiment are more convenient to use, although more expensive. These are available in a wide variety of sizes, layer thickness, stationary phase material, activity, inert support, etc. For routine use silica gel plates coated on sheets of inert plastic are very convenient. Such pre-coated plates will be used in this course. For rapid qualitative analysis small TLC plates about the size of microscope slides are particularly useful. 8 The TLC plate is prepared for development by introducing very small amounts of the mixtures to be analyzed near one end of the plate, usually about 5-10 mm or so from one end. During the development, the end of the plate below the initial spotting points will be immersed in a shallow pool of the mobile phase material, and it is important to position the spots high enough on the TLC plate so that they will not be below the surface of the mobile phase. Spots tend to spread in all directions by diffusion during development, and the initial spots should be far enough apart that they will not run together when this occurs. This also means that the size of the initial spots should not be too large. Spots should not be too close to the sides of the plate, since movement of the mobile phase during development is often not uniform near the edges of the plate. On microscope slide-sized plates it is usually possible to spot three or four samples side-by-side on the same plate. The "Before" plate in Figure 1 shows how a plate might look after three samples, 1, 2, and 3 had been spotted side-by-side. Figure 1. TLC Plate Before and After Development Spotting the sample is done by preparing a solution of the sample in a convenient volatile solvent (acetone, dichloromethane are common) and then transferring a few microliters of this solution to the plate. This may be done using a micropipette, which can be prepared from a melting point capillary tube. A region about 1-2 cm wide in the center of the capillary tube is softened in a Bunsen burner flame (hold the tube just above the bright blue cone of the flame and rotate the tube so that the portion being heated is softened uniformly). The capillary tube is then removed from the flame, and the softened part is immediately drawn to a very narrow tube by pulling out on both ends of the tube. This may require some practice, but is very easy once you learn how. Break the capillary in the middle of the narrow part and also break off the original closed end of the melting point tube to give two micropipettes. The micropipette is filled with the solution to be spotted by dipping the end below the surface of the sample solution, so that some of the solution is drawn into the tube by capillary action. The end of the filled micropipette is then touched to the surface of the TLC plate at the point where you wish to position the spot, and a little of the liquid runs out of the pipette onto the plate. It is desirable to keep the diameter of the original spot as small 9 as possible, so instead of touching the pipette to the plate and leaving it in contact until the pipette is empty, the pipette is touched only briefly to the plate. This is repeated as many times as necessary at the same point, letting the solvent evaporate from the plate between touches, until enough material has been deposited for good results. It is not always obvious when enough material has been deposited, particularly if the components of the mixture are all colorless. In those cases it may be necessary to use a bit of trial and error until satisfactory results are obtained. Once the sample has been introduced onto the plate, the chromatogram is developed by placing it upright in a developing chamber that contains some of the mobile phase liquid a few millimeters in depth. The atmosphere inside the developing tank must be kept saturated with the vapor of the mobile phase in order to get reproducible results. Typically a "wick" of porous paper such as filter paper is used to partially line the inside of the tank and keep the atmosphere in the tank saturated with the mobile phase vapor. Only the very bottom edge of the plate, below the level of the spots is immersed in the liquid. The mobile phase rises up the plate through capillary action until the solvent front has moved an appropriate distance. If you wish the solvent front to move a pre-determined distance, you may score a line across the plate at that point using a spatula blade; this interrupts the continuity of the stationary phase layer, and the mobile phase will stop moving when it reaches the scored line. In other cases you may simply let the solvent front run all the way to the upper end of the plate. The "After" diagram in Figure 1 shows the appearance of a plate after development is complete. The plate is removed from the tank, the solvent is allowed to evaporate, and the chromatogram is read. To read the thin layer chromatogram one marks the center point of each spot. This is easy if the components are colored and readily visible, but if some of the components cannot be directly detected visibly, some treatment may be needed first so that they can be seen. A very useful method is to expose the developed and dried plate to iodine vapor in a sealed vessel (e.g., a jar containing a few crystals of iodine). For many organic compounds, iodine will be reversibly and selectively adsorbed on the plate at points where the spots of organic material are located, producing readily visible brown spots. These spots usually will fade away after the plate is removed from the iodine jar, so their positions should be marked before this happens. Sometimes a fluorescent substance is incorporated in the stationary phase layer, and the positions of spots can be seen by examining the plate under ultraviolet light. There are other ways the spots may be detected, depending on the substances being examined. Once the positions of the spots has been established, measurements are made from the initial spotting point (the base line) to the centers of the spots and from the initial spotting point to the position of the solvent front when development was complete. In Figure 1 three samples have been analyzed. Sample 1 gives only a single spot, which has moved a distance X from the original spotting point, when the solvent front had traveled a distance M, again measured from the point where the initial spots were introduced not from the lower edge of the plate. Sample 3 also gives a single spot, but it has moved a distance Y. Sample 2 gives two spots, one of which has moved distance X, and the other distance Y. One records the data from the chromatogram in terms of the ratios of the distances traveled by each component compared to the distance traveled by the solvent. This is expressed as the "Rf" value for each of the components. The term "Rf" is the "ratio to front", or the ratio of the distance 10 traveled by a compound to the distance traveled by the mobile phase solvent during development. Rf values are calculated as shown below. For substance in sample 1: Rf X/M For substance in sample 3: Rf Y/M The Rf value for a compound is reproducible if the conditions of analysis can be duplicated exactly. The results for