2 - Thin Layer Chromatography

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   Experiment 2  –   Chem 276  –   Fall Semester 2010 Page 1  of 5   Experiment 2 - Thin Layer Chromatography Introduction Chromatography is an experimental technique by which a mixture of compounds can be separated into its individual components. In thin layer chromatography, there is a stationary phase as well as a mobile phase. For this experiment, the TLC plate consists of an aluminum backing on which is placed a thin layer of silica gel. The silica gel is the stationary phase. The eluding solvent is the mobile phase which carries with it the components of the mixture. Thin layer chromatography is used mostly for analytical  purposes. Microgram to picogram quantities can be separated by this technique and then characterized by their R  f   value. The retention factor, or R  f  , is defined as the distance traveled by the compound divided  by the distance traveled by the solvent. Rf =distance traveled by compounddistance traveled by solvent fro  In the example shown below, a compound travels 16.2 cm while the solvent front travels 23.0 cm, the R  f   is 0.704: Rf =solvent frontsrcinnew positionof compound 16.2 cm23.0 cm16.223.0 = 0.704  The R  f   for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant:    solvent system    adsorbent    thickness of the adsorbent    amount of material spotted    temperature   Experiment 2  –   Chem 276  –   Fall Semester 2010 Page 2  of 5  Since these factors are difficult to keep constant from experiment to experiment, relative R  f    values are generally considered. “Relative R  f  ” means that the values are reported relative to a standard, or it means that you compare the R  f   values of compounds run on the same plate at the same time. The larger an R  f   of a compound, the larger the distance it travels on the TLC plate. When comparing two different compounds run under identical chromatography conditions, the compound with the larger R  f   is less polar because it interacts less strongly with the polar adsorbent on the TLC plate. Conversely, if you know the structures of the compounds in a mixture, you can predict that a compound of low polarity will have a larger R  f   value than a polar compound run on the same plate. The R  f   can provide corroborative evidence as to the identity of a compound. If the identity of a compound is suspected but not yet proven, an authentic sample of the compound, or standard, is spotted and run on a TLC plate side by side (or on top of each other) with the compound in question. If two substances have the same R  f   value, they are likely (but not necessarily) the same compound. If they have different R  f values, they are definitely different compounds. Note that this identity check must be performed on a single plate, because it is difficult to duplicate all the factors which influence R  f   exactly from experiment to experiment.   Experiment 2  –   Chem 276  –   Fall Semester 2010 Page 3  of 5   Procedure 1.   Cut six 1½ × 4 inch pieces of TLC plates. 2.    Next you will need to draw a line 1 cm from the bottom of your TLC plates with a pencil. See diagram in introduction. 3.   To one plate, spot on the line with any two of the five compounds listed in Table 1. The spots should be about 1 cm apart and on the line. To insure that your plate is properly spotted use an UV light. Remember the identity of each spot. 4.   To another plate, spot on the line with any two remaining compounds listed in Table 1. The spots should be about 1 cm apart on the line. To insure that your plate is properly spotted use an UV light. Remember the identity of each spot. 5.   To final plate, spot on the line with final compound from Table 1 and the unknown mixture. The spots should be about 1 cm apart on the line. To insure that your plate is properly spotted use an UV light. Remember the identity of each spot. 6.   Prepare the following eluding solvents in separate 100 mL beakers. A)   Prepare a 1:1 mixture of ethyl acetate and hexanes. Do this by mixing 25 mL of ethyl acetate with 25 mL of hexanes. B)   Prepare a 1:4 mixture of ethyl acetate and hexanes. Do this by mixing 10 mL of ethyl acetate with 40 mL of hexanes. 7.   Pour enough of the1:1 mixture of eluding solvent in three separate developing chambers until the depth is approximately ½ cm. Be sure that the solvent surface is below the application points. (i.e. The spots should not be in the solvent.) 8.   Place each TLC plate in a developing chamber containing the 1:1 mixture of ethyl acetate and hexanes and allow the solvent to climb the plate until in comes with in a ½ cm of the top of the plate. Once this occurs, remove the  plates from the chambers and mark the solvent fronts with a pencil. 9.   Allow the TLC plates to dry. 10.   Take the TLC plates to a dark room and with the use of a UV light circle the illuminated spots with a pencil. 11.   Stain your TLC plate by dipping it in a stain solution and drying over heat gun as directed by your TA. 12.   Calculate the R  f   values for each of the spots. 13.   Repeat steps 7 thru 12 with the 1:4 ethyl acetate and hexanes eluding solvent. 14.   Dispose of used TLC plates and elution solvent as directed by your TA. Table 1 Acetophenone Anisole Benzoic Acid Benzyl Alcohol Benzaldehyde   Experiment 2  –   Chem 276  –   Fall Semester 2010 Page 4  of 5   Questions 1.   Draw the structures of the following compounds. Acetophenone Anisole Benzoic Acid Benzyl Alcohol Benzaldehyde Compound R   f   in 1:1 EtOAc/Hex R   f   in 1:4 EtOAc/Hex Appearance in  ___________ stain Appearance in  __________ stain Acetophenone Anisole Benzoic Acid Benzyl Alcohol Benzaldehyde 2.   Calculate the R   f values of each spot.
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