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Thin Layer Chromatography (TLC) Tutorial

Key Concepts

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TLC Technique

  1. Application of the Sample

    1. 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.

    2. 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.
    3. The spot is dried.
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  2. Development

    1. 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.
    2. 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.
    3. The mobile phase solvent will rise slowly up the TLC plate by capillary action.
    4. 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).
    5. 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, Rf

    (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, Rf, for the blue spot:

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

    We can calculate the retardation factor, Rf, 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, Rf 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 Rf (closer to 0)

    • large distance travelled = large Rf (closer to 1)

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Relative Solubility in the Solvent and Adsorption to the Stationary Phase

The further up the TLC plate a component spot moves, the more soluble it is in the mobile (solvent) phase and the less strongly it adsorbs or binds to the stationary phase.

more soluble in mobile (solvent) phase Your browser does not support HTML5 canvas. adsorbs least to stationary phase
least soluble in mobile (solvent) phase adsorbs most to stationary phase

The TLC plates you use will usually have silica gel as the stationary phase.

Silica gel is a form of silicon dioxide (SiO2, silica), a three-dimensional covalent network.

At the surface of the silica gel, there are oxygen atoms that have formed only one covalent bond to a silicon atom. These oxygen atoms will covalently bond to hydrogen atoms forming a polar -OH bond.

The silica gel can therefore attract molecules using the stronger hydrogen bonds or dipole-dipole interactions as well as the weak intermolecular forces (london forces or dispersion forces).

Polar substances will adsorb, or bind, to the surface of the silica gel more strongly using hydrogen bonds or dipole-dipole interactions (as well as the weak intermolecular forces).

Non-polar substances will adsorb weakly to the surface of the silica gel using only the weak intermolecular forces (london forces or dispersion forces).

Hδ+Hδ+Hδ+
///
Oδ-
|
Oδ-
|
Oδ-
|
-O-Si-O-Si-O-Si-O-
|||
silica gel on TLC plate

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:

If we use a polar solvent as the mobile phase:

Polarity of Components in a Mixture
polar componentnon-polar component
c
o
n
d
i
t
i
o
n
s
polar stationary phase
non-polar mobile phase
Adsorbs strongly to TLC plate.
Less soluble in solvent.
Spot travels smallest distance.
Smaller Rf
Adsorbs weakly to TLC plate.
More soluble in solvent.
Spot travels furthest distance.
Larger Rf
non-polar stationary phase
polar mobile phase
Adsorbs weakly to TLC plate.
More soluble in solvent.
Spot travels furthest distance.
Larger Rf
Adsorbs strongly to TLC plate.
Less soluble in solvent.
Spot travels smallest distance.
Smaller Rf

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