Thin Layer Chromatography (TLC) Chemistry Tutorial

Key Concepts

• Thin Layer chromatography (TLC) is an analytical technique used to separate a mixture into its components.
• The process of thin layer chromatography is similar to paper chromatography but thin layer chromatography:

  (a) is usually faster

  (b) achieves better separations
  (due to greater choice in stationary and mobile phases)

• The stationary phase is a usually a thin layer of silica gel or alumina.
• The stationary phase is coated onto a piece of glass, metal or plastic to make it rigid.
• The combination of the glass (metal or plastic) and the stationary phase is referred to as the thin layer chromatography plate (TLC plate).
• The mobile phase refers to the solvent that moves up the TLC plate by capillary action.
• Components in a mixture are separated based on their different abilities to bind or adsorb to the stationary phase, and on their different abilities to desorb or dissolve in the mobile solvent phase.
• The retardation factor, R_f, is a comparison of the distance travelled by a component to the distance travelled by the mobile solvent.

  Rf =	distance component travelled distance solvent travelled

• R_f values depend on temperature, composition of the TLC plate used and the composition of the solvent used.
  For these reasons, it is usual to run samples of known substances at the same time as you run the sample of the unknown mixture.

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TLC Technique

1. Application of the Sample

   A starting point is marked on the TLC plate using pencil, shown on the diagram as a grey line. Do not use a pen containing an ink that will be soluble in the mobile solvent phase. A spotter, such as a thin capillary tube, is used to place a very small spot of the solution mixture on the TLC plate. Shown as a black dot on the diagram. The spot is dried.

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2. Development

   The TLC plate is placed in a vessel containing a small amount of the mobile phase (the solvent). The starting point marked on the TLC plate must be above the line of the solvent. A lid or cover is placed over the vessel. This ensures that the atmosphere in the vessel is saturated with the solvent vapour which stops the solvent from evaporating as it rises up the TLC plate. The mobile phase solvent will rise slowly up the TLC plate by capillary action. The components of the mixture to be separated will also rise up the TLC plate, but at a slower rate than the mobile phase. Components with high solubility in the mobile solvent phase but low adsorbance to the stationary phase move up the TLC plate quickly. (blue spot). Components with low solubility in the mobile solvent phase and high adsorbance to the stationary phase move slowly up the TLC plate. (olive spot). When the solvent front (the level of the mobile solvent phase) has almost reached the top of the TLC plate, the position of the solvent front is marked and the TLC plate set aside to dry.

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3. Detection

   If the components are different colours, you can see the position of each component on the TLC plate.

   If the components are colourless, there are a number of techniques used to detect each component:

   • Reactions with colour producing reagents:

     Spraying colourless amino acid spots with ninhydrin produces visible purple spots.
     Exposing the TLC plate to iodine vapour shows the components up as brownish spots.

   • Placing the sample under ultraviolet light (if the sample fluoresces or absorbs ultraviolet light).

     The position of each component must be marked on the TLC plate before turning off the UV light.

   • Often a substance is added to the stationary phase which will fluoresce when exposed to ultraviolet light.

     When UV light shines on this TLC plate, the separated components of the mixture will appear as non-glowing spots on a glowing background.
     The position of each component is marked on the TLC plate before turning off the UV light.

4. Calculating the Retardation Factor, R_f

   (i) Measure the distance between the starting point (origin) and the solvent front using a ruler. On the TLC plate shown the distance is shown in black. The solvent has travelled 25 mm.

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   (ii) Measure the distance between the starting point (origin) and the blue spot using a ruler. On the TLC plate shown the distance is shown in blue. The blue spot has travelled 18 mm.

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   (iii) Calculate the retardation factor, R_f, for the blue spot:

   Rf =	distance blue spot travelled distance solvent travelled
   =	18 mm 25 mm
   =	0.72

   We can calculate the retardation factor, R_f, of the other two spots in the same way:

   component	distance travelled (mm)	Rf = distance spot travelled distance solvent travelled
   solvent	25
   blue spot	18	Rf = 18 25 = 0.72
   purple spot	13	Rf = 13 25 = 0.52
   olive spot	5	Rf = 5 25 = 0.20

   Check that your calculations make sense!

   Retardation factors, R_f values, must be less than 1 (a component can't travel faster than the solvent!).
   The further a spot has moved up the TLC plate the larger the value of its retardation factor:

   
   
   • small distance travelled = small R_f (closer to 0)
   
   
   • large distance travelled = large R_f (closer to 1)

Relative Solubility in the Solvent and Adsorption to the Stationary Phase

As soon as the solvent front reaches the place where we have placed our spot of mixture, the components in the mixture will dissolve in the solvent.
How well each component dissolves in the solvent depends on factors such as the relative polarity of the solvent molecules and the molecules in the mixture.

If we use a non-polar solvent as the mobile phase:

• non-polar components in the mixture will dissolve more easily.
• polar components in the mixture will dissolve less easily.

If we use a polar solvent as the mobile phase:

• polar components in the mixture will dissolve more easily.
• non-polar components in the mixture will dissolve less easily.