CHM 220   Thin Layer Chromatography

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


Thin-layer chromatography (TLC) is a simple and inexpensive analytical technique that can quickly and efficiently separate quantities of less than ten micrograms of material.  TLC has many applications in the organic laboratory.  TLC is used for the rapid analysis of reagent and product purity, or to quickly determine the number of compounds in a mixture.  Also, by comparing an unknown compound’s behavior to the behaviours of known standard compounds, mixture compounds can be tentatively identified. 

Chemistry frequently use TLC to follow the progress of a reaction by monitoring the disappearance of a reactant or the appearance of a product.  Also TLC often is used to select a suitable solvent before attempting a larger scale column chromatography separation.  Then, during the column chromatography experiment, TLC is frequently used to monitor the separation. 

The term chromatography refers to several related techniques for analyzing, identifying or separating mixtures of compounds.   All chromatographic techniques have a two-part operation in common.  In each technique a sample mixture is placed into a liquid or gas, called a mobile phase.   The mobile phase carries the sample through a solid support, called the stationary phase, which contains an adsorbent or another liquid.   The different compounds in the sample mixture move through the stationary phase at different rates, due to the different attractions for the mobile an stationary phases.  Thus, individual compounds in the mixture separate as they move through the stationary phase.  The separate compounds can be collected or detected, depending on the particular chromatographic technique involved. 

In, TLC, capillary action allows a liquid (mobile phase) to ascent a solid (stationary phase) coated on a support plate.  A sample of the compound mixture is applied near the bottom of a dry TLC plate, as shown in Figure 1(a). 

The plate is placed into a developing chamber, a covered container with a shallow layer of mobile phase liquid in the bottom (see Figure 2).  As the mobile phase ascends the plate, the mixture compounds dissolve in the mobile phase to different extents, due to the differences in their relative attractions for the mobile and stationary phases.  After the separation si complete, the TLC plate is called a chromatogram, as shown in Figure 1(b). 

Figure 2. Developing Chambers

During the TLC process, the solid stationary phase, called the adsorbent, adsorbs the mixture compounds.  As the mobile phase, called the eluent, travels up over the adsorbent, the compounds within the mixture move at different rates.  A reversible and continuous competitive attraction between the eluent and the adsorbent for the mixture compounds causes this rate difference. 

Compounds with less attraction for the adsorbent move rapidly with the eluent.  Compounds with the more attraction for the adsorbent move slowly with the eluent.  Because TLC adsorbents are typically very polar, the more polar is a compound in the mixture, the more strongly it adheres to the adsorbent and the more slowly it moves. 

Similarly, intermolecular attractions between the eluent and the compounds determine the solubility of the compounds in the mobile phase.  In general, the more polar the eluent, the more rapidly agiven compound moves.  Polar compounds, which are strongly attracted to the adsorbent, require polar eluents to attract them away from the adsorbent. 

TLC plate development sequence:

  1. a mixture of a red and blue compound is separated as the plate develops, i.e. as the light blue solvent moves up the plate.
  2. Once visible, the Rf value, or Retention factor, of each spot can be determined by dividing the distance traveled by the product by the total distance traveled by the solvent (the solvent front).
    1. These values depend on the solvent used, and the type of TLC plate, and are not physical constants.
  3. The appropriate solvent in context of TLC will be one which differs from the stationary phase material in polarity.
  4. If polar solvent is used to dissolve the sample and spot is applied over polar stationary phase TLC, the sample spot will grow radially due to capillary action, which is not advisable as one spot may mix with the other.
  5. To restrict the radial growth of sample-spot, the solvent used for dissolving samples in order to apply them on plates should be as non-polar or semi-polar as possible when the stationary phase is polar, and vice-versa.   See Table 1 for the approximate order of polarity of eluents used in chromatography. 

Determining a Retention Factor, Rf

The ratio of the distance that a compound moves to the distance that the eluent front moves is called the retention factor, denoted as Rf (see equation 1). For example, in Figure 2, the stock sample compound moved distance A while the eluent front traveled distance S.  If distance A is 25 millimeters (mm) and distance S is 55 mm, then the Rf is calculated as

The chromatographic behavior or individual compounds is reproducible as long as the stationary and mobile phases and the temperature are kept constant.  Therefore, an Rf can be used for identification purposes. 

When a compound is strongly attracted to the adsorbent and does not travel very far from the origin, or point of application, the Rf is small.  An increase in eluent polarity would probably increase the attraction of the compound for the eluent.  As a result, the compound would move farther up the plate, resulting in a larger Rf. 

Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase.

  1. if normal phase silica gel is used as the stationary phase it can be considered polar.
  2. Given two compounds which differ in polarity, the most polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places.
  3. Consequently, the less polar compound moves higher up the plate (resulting in a higher Rf value).
  4. If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plate.
  5. Practically this means that if you use a mixture of ethyl acetate and heptane as the mobile phase, adding more ethyl acetate results in higher Rf values for all compounds on the TLC plate.
  6. Changing the polarity of the mobile phase will not result in reversed order of running of the compounds on the TLC plate. If a reversed order of running of the compounds is desired, an apolar stationary phase should be used, such as C18-functionalized silica.

TLC Example - Chromatogram of 10 essential oils colored with vanillin reagent.

Examples of common problems encountered in TLC:

  • The compound runs as a streak rather than a spot – The sample was overloaded.
    • Run the TLC again after diluting your sample. Or, your sample might just contain many components, creating many spots which run together and appear as a streak. Perhaps, the experiment did not go as well as expected.
  • The sample runs as a smear or a upward crescent.
    • Compounds which possess strongly acidic or basic groups (amines or carboxylic acids) sometimes show up on a TLC plate with this behavior. Add a few drops of ammonium hydroxide (amines) or acetic acid (carboxylic acids) to the eluting solvent to obtain clearer plates.
  • The sample runs as a downward crescent.
    • Likely, the adsorbent was disturbed during the spotting, causing the crescent shape.
  • The plate solvent front runs crookedly.
    • Either the adsorbent has flaked off the sides of the plate or the sides of the plate are touching the sides of the container (or the paper used to saturate the container) as the plate develops. Crookedly run plates make it harder to measure Rf values accurately.
  • You see a blur of blue spots on the plate as it develops.
    • Perhaps, you used an ink pen instead of a pencil to mark the origin?

Reference:Adapted from Modular Laboratory Program in Chemistry Tech 707 by Joe Jeffers