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Lab 707 Separating a Mixture of Biphenyl, Benzhydrol, and Benzophenone by Thin- Layer Chromatography


The purpose of this experiment is to figure out a proper solvent that will be used to separate a mixture of biphenyl, benzhydrol, and benzophenone by thin-layer chromatography. The thin-layer chromatography will be used to investigate solvent polarity on relative compounds in the mixture, and to identify the specific compounds in an unknown mixture containing any combination of biphenyl, benzhydrol, and benzophenone. Each separate compound will have an R f value that will be calculated in order to compare the combinations of the unknown to those of separate compounds in the stock solution. The student should already know how to use a Bunsen burner or microburner. Thin-layer chromatography (TLC) is a simple and cheap technique that can efficiently separate quantities of less than ten micrograms of material. TLC is used for the rapid analysis of reagent and product, purity, or to determine the number of compounds in a mixture. It’s also used to compare unknown compounds behaviors to those of known compounds so that the unknown compound can be broken down to what is in the mixture. The term chromatography refers to several related techniques for identifying, separating, or analyzing mixtures of compounds. All chromatographic techniques have a two part operation in common. The first is the mobile phase, where a sample mixture is placed in a liquid or gas. The second is the stationary phase, where the mobile phase carries the sample through a solid support. The different compounds in the sample mixture move through the stationary phase at different rates because of the difference in reactions between the mobile and stationary phases. This way each compound in the mixture will be at a different location when the chromatography is done. During the TLC process, the solid stationary phase is called the adsorbent. As the mobile phase (eluent) travels up the adsorbent the compounds within the mixture will begin to separate. Compounds with less attraction for the absorbent move rapidly with the eluent. Because TLC adsorbents are typically polar, the more polar a compound is in the mixture the stronger it adheres to the adsorbent and the slower it moves. The ratio of the distance that a compound moves to the distance that the eluent front moves is called the retention factor (R f ). The equation for R f is:

R f = distance traveled by compound (mm)/ distance traveled by eluent front (mm)


1) Preparing the Developing Chamber

Obtain 5 250-ml beakers and label each beaker with one of the eluets: ethyl acetate, hexane, methanol, dichloromethane, and toluene. Obtain 5 rubber bands and pieces of plastic large enough to cover the top of the beakers. Cut a piece of filter paper into a rectangle wide enough so extends nearly to the top and long enough so it wraps around ¾ of the beaker. Do this for all five beakers. Pour

5 ml of the appropriate solvent into its beaker and cover each beaker with the plastic and set it aside.

2) Spotting the TLC Plates

Get five 2.5x7.5 cm TLC silica gel plates and be very careful to make sure the coated surface of the TLC plates are not touched. Using a pencil, lightly label the top of each plate with the name of one of the five solvents. Mark the origin on each of the five plates lightly by drawing a line across the bottom. Also, mark two cross hatch lines on the bottom line to indicate where the solution will be spotted.

Caution: benzhydrol, benzophenone, and biphenyl in acetone solutions are flammable and irritating. Avoid using these compounds near heat sources and prevent any contact. Also, avoid inhaling vapors or ingesting these compounds.

Obtain a vial of the stock solution mixture containing all three compounds. Place the drawn out end of a micropipette into the stock solution and allow the liquid to rise up the capillary. Spot the solution onto the TLC plate by quickly and lightly touching the end of the pipette. The spot should be less than 2 mm. Allow the solvent to completely dry, and then make a second spot on one of the hatch marks. Allow the solvent to evaporate again. Make sure to do this for all five plates.

3) Developing TLC Plates

Check to make sure the eluent level in the chambers is below the line on the TLC plates. Place each spotted TLC plate into its appropriate chamber using the chamber wall to support the plate. Cover the chambers with the plastic wrap and secure it with the rubber band. When the eluent rises to about 1cm under the top of the plate, remove the plate from the chamber and immediately mark a light line where the eluent front was.

Caution: ultraviolet radiation can cause severe damage to eyes. Wear goggles and do not look directly into the UV lamp.

When all five plates are done, take them out of their chambers and allow them to dry off. Take the plates over to the UV light and have them examined. Look for the eluent that gave three separate spots for the mixture, this will be your solvent. Circle the spots on the TLC papers.

4) Analyzing an Unknown Mixture

Obtain an unknown solution and 3 new TLC plates. Each plate should have two hatch marks for spotting. Label each of the hatch marks with a separate solution; stock, benzhydrol, benzophenone, biphenyl, and an unknown. Place a drop of each of the solutions to the corresponding hatch mark. Place each of the TLC into the eluent that was chosen from the previous step. Make sure the TLC plates are supported against the beaker walls and if there is more than one TLC plate in the beaker at a time, that the

plates never touch each other. When the eluent of each of plates rises to about 1 cm under the top of the plate, remove it and allow it to dry. When all three plates are done take them to the UV lamp and examine them. Circle all spots with a pencil. The stock solution should have three spots, one for biphenyl, benzhydrol, and benzophenone. Line up the spots from the unknown mixture to those of biphenyl, benzhydrol, and benzophenone. Calculate the R f values for all the spots and record the compounds present in the unknown solution.


None of the eluets separated the stock solution into three separate compounds except for toluene. The TLC plates for the unknown analysis were marked as follows:

Stock Benzophenone
Benzhydrol Biphenyl

Each eluent front went 70 mm.

R f Benzophenone = 30mm/70mm = 0.43mm R f Benzhydrol = 15mm/70mm = 0.21mm R f Biphenyl = 60mm/70mm = 0.86mm

unknown 9 unknown 4
unknown 9
unknown 4

R f unknown 9 = 35mm/70mm = 0.50mm R f unknown 4 = 15mm/70mm, 30mm/70mm, 60mm/70mm = 0.21mm, 0.43mm, 0.86mm


The lab at first did not go well. None of the eluets were breaking the stock solution into three separate compounds. Finally after 3 trials, toluene was shown to be the proper solvent. The results came out smoothly after that. The unknown 9 solution was made of Benzophenone, and the unknown 4 solution had all three compounds.