CHM 220 Lab  |  Thin Layer Chromatography 

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

Purpose of the Experiment

  • Select a solvent to separate a mixture of biphenyl, benzhydrol, and benzophenone by then-layer chromatography. 
  • Identify the mixture compounds by comparing Rf values with reference compounds. 
  • Use thin-layer chromatography to investigate solvent polarity effects on the relative motilities of these compounds in a mixture. 
  • Use thin-layer chromatography to identify the specific compounds in an unknown mixture containing any combination of biphenyl, benzhydrol, and benzophenone. 


Using TLC in an Experiment – A TLC experiment has three general stages:  spotting, developing, and visualizing. 

  1. Spotting a Plate:  The origin is marked, usually by drawing a thing line across the bottom of the plate with a pencil, as show in in Figure 3. 

    The sample should be dissolved in a volatile solvent such as acetone or dichlormethane.  A glass capillary tube is used to apply a small amount of sample solution onto the plate, keeping the sample in as small an area as possible.  With practice, spots with diameters of 1-2 mm can be produced
    After the solvent evaporates, additional sample solution can be applied to the sample spot.  Application of too much sample can lead to “tailing” and poor evaporation, as shown in Figure 3. 
  2. Developing a Plate: When the spotted plate is placed into the chamber, the origin marked on the plate must be higher than the level of the eluent, to prevent the sample from dissolving from the plate into the eluent layer.  When the eluent reaches a point approximately 10 mm from the top of the plate, the plate is removed from the chamber.  The point that the eluent has reached is called the eluent front and is immediately marked with a pencil (see Figure 3).  The plate is dried by allowing the eluent to evaporate form the plate. 
    If the eluent front is allowed to reach the top of the plate, the mixture compounds may continue to move along the plate.  An Rf  measurement under these circumstances is NOT valid. 
    Optional:   A piece of filter paper can be placed along the walls of the developing chamber, which contains a shallow layer of the appropriate eluent.  The paper acts as a wick that adsorbs the eluent and ensures that, then the chamber is closed; its atmosphere is saturated with eluent vapor, minimizing evaporation from the plate. 
  3. Visualizing the Compound – Upon development, a successful separation of colored compounds will reveal distinct spots, indicating that the mixture compound have separated (see Figure 3).  To make separated colorless compounds observable to the eye, the spots are treated in some way to make them visible.  The process is called visualization. 
    Some compounds fluoresce.  Such compounds can be visualized by viewing the TLC plate under an ultraviolet (UV) lamp.  Frequently, the adsorbent contains a chemically inert fluorescent material.  When viewed under UV light, compounds that adsorb the UV light appear as dark spots that may be outlined with a pencil. 
    Another simple method for visualizing organic compounds is to place the chromatogram in a chamber containing iodine crystals and vapor.  The I2 vapor forms a colored complex with many compounds and allows detection of their spots.  The spot location must be marked immediately because the I2 will eventually sublime from the plate. 


  • 5 beakers, 250 mL, with plastic wrap to cover each
  • 12cm filter paper, cut to fit developing chamber
  • Glass stirring rod
  • Gloves
  • 10mL graduated cylinder
  • Labels and marking pens
  • 5-6 micropipet capillary tubes
  • Rulers
  • 9 TLC plates, silica gel, 2.5 x 7.5 cm with fluorescent indicator. 
  • Reagents:  benzhydrol, benzophenone, biphenyl, iodine
  • Solvents:  dichloromethane, ethyl acetate, hexane, methanol, toluene

Procedure:  Please conduct the experiments in the fume hood.  Link to Data Sheet

  1. Preparing the Developing Chambers:  Obtain five 250ml beakers and label each beaker with the name of one of the eluents:  ethyl acetate, hexane, methanol, dichlormethanke, and toluene.  Obtain 5 rubber bands and five pieces of plastic wrap, each large enough to cover the beaker.
    Cut a piece of filter paper into a rectangle wide enough so that the paper extends nearly to the top of the TLC plate and only long enough so that the paper covers three quarters of the beaker wall (see Figure 4).  Cut papers to fit the other four beakers. 
    Using a glass stirring rod to direct the flow, pour 5 mL of the appropriate eluent into each labeled beaker to moisten the filter paper liner and to form a layer 3-4 mm deep.  Cover each developing chamber with plastic wrap and set aside. 
  2. Spotting the TLC plates:  Obtain five TLC silica gel plates.  Avoid touching the coated surface of the plate with your fingers.  Hold the plate at the top or by the sides.  Use a PENCIL to mark on the TLC plate (Inks dissolve in the eluents).  With a pencil, label each plate at the top with the name of one of the five eluents. 
    Mark the origin on each plate by making a very faint pencil line across the place 1 cm from the bottom (the pencil lead can cut completely through the adsorbent, forming a gap that may stop the flow of eluent).  Faintly mark two cross-hatch lines on the origin line to indicate where the solution will be spotted (see Figure 1a in the introduction). 
    Obtain a vial of the stock solution mixture containing biphenyl, benzhydrol, and benzophenone.  Place the micropipette into the stock solution and allow the liquid to rise by capillary action.  Spot the solution onto one TLC plate by quickly and lightly touching the end of the micropipette to the surface of the adsorbent at each corss-hatch.  Transfer an amount of liquid to the plate that produces a spot with a diameter less than 2mm.  Allow the solvent to dry completely between applications to the same spot.  Otherwise, the spot will become too large.  Note:  use a new micropipette for each solution. 
    After the sample solvent has evaporated, make a second application to one of the two spots to increase the sample amount.  Allow the solvent to evaporate. 
  3. Developing TLC Plates:  Check to be certain that the eluent level in each developing chamber is BELOW the point where the samples have been spotted.  Place the spotted TLC plate into the ethyl acetate developing chamber.  Use the chamber wall to support the plate as shown in Figure 4.  Avoid leaning the plate against the filter paper.  Eluent on the filter paper can be absorbed by the adsorbent on the plate and interfere with the ascending eluent.
    Cover the chamber with a piece of plastic wrap, secured with a rubber band. 
    When the eluent front rises to within 1 cm of the top of the plate, remove the plate from the chamber and IMMEDIATELY mark the eluent front with a pencil.  Allow the eluent to evaporate from the plate under a fume hood. 
  4. Examine the developed TLC plate under an ultraviolet light.  Use a pencil to circle any visualized spots. 
  5. Using each of the four other eluents and new TLC plates, repeat the procedures for spotting the stock solution and developing the chromatogram.  Use the same micropipette to spot the stock solution on each plate. 
  6. Compare the chromatograms from each eluent chamber.  Compare the spots from both single and double sample applications.  Record the name of the eluent that gives the best separation of the mixture.  Use this eluent in Parts 7 and 8.  Record whether a single sample application or a double sample application gives better results. 
    Use a ruler to measure the distance from the origin to the center of each spot on the plate developed with the chosen eluent.  Measure the distance from the origin to the eluent front.  Use Equation 1 to calculate the Rf for each spot. 
  7. Identifying the Compounds in a Mixture:  Obtain individual solutions of each component in the mixture.  Using a new micropipette for each new solution, spot each one alongside the stock solution mixture on a new TLC plate.  Develop the plate in the eluent identified in Part 6 that gives the most efficient separation.  Visualize the chromatogram using UV light.  Use a pencil to outline the spots.  Calculate the Rf for each compound.  Record each Rf and the identity of each compound.

    Place one of the developed chromatograms into an I2 chamber prepared by the instructor.  Allow the chromatogram to remain in the chamber for 5 minutes.  Describe the appearance of the plate after visualization with I2. 
  8. Analyzing an Unknown Mixture:  Obtain an unknown solution and a new TLC plate.  Spot the unknown solution and the stock solution mixture onto the plate.  Develop the plate in the eluent identified in Part 6.  Visualize the spots with either UV light or I2.  Calculate Rfs for each compound in the mixture.  Record the compounds present in the unknown solution. 
  9. Cleaning Up:  Pour any remaining eluent into the appropriate waste containers in the hood.