Thin Layer Chromatography, TLC (OCR A Level Chemistry)

• Thin-layer chromatography (TLC) is a technique used to analyse small samples via separation
  • For example, we could separate a dye out to determine the mixture of dyes in a forensic sample

• There are 2 phases involved in TLC:
  • Stationary phase
    • This phase is commonly thin metal sheet coated in alumina (Al₂O₃) or silica (SiO₂)
    • The solute molecules adsorb onto the surface
    • Depending on the strength of interactions with the stationary phase, the separated components will travel particular distances through the plate
    • The more they interact with the stationary phase, the more they will 'stick' to it

  • Mobile phase
    • Flows over the stationary phase
    • It is a polar or non-polar liquid (solvent) that carries components of the compound being investigated
    • Polar solvents - water or alcohol
    • Non-polar solvents - alkanes

• If the sample components are coloured, their spots are easily identifiable on the chromatogram
• If the sample components are not coloured, then we can locate the spots on the chromatogram and draw around them in pencil
  • To locate the spots we can use:
    • UV light
    • Ninhydrin (carcinogenic)
    • Iodine vapour

Conducting a TLC analysis

• Step 1:

  Prepare a beaker with a small quantity of solvent

• Step 2:

  On a TLC plate, draw a horizontal line at the bottom edge (in pencil)

  This is called the baseline
• Step 3:
  Place a spot of pure reference compound on the left of this line, then a spot of the sample to be analysed to the right of the baseline and allow to air dry

  The reference compounds will allow identification of the mixture of compounds in the sample

• Step 4:

  Place the TLC plate inside the beaker with solvent - making sure that the solvent does not cover the spot - and place a lid to cover the beaker

  The solvent will begin to travel up the plate, dissolving the compounds as it does
• Step 5:
  As solvent reaches the top, remove the plate and draw another pencil line where the solvent has reached, indicating the solvent front

  The sample’s components will have separated and travelled up towards this solvent front

A dot of the sample is placed on the baseline and allowed to separate as the mobile phase flows through the stationary phase; The reference compound/s will also move with the solvent

R_f values

• A TLC plate can be used to calculate R_f values for compounds

R_f = 

• These values can be used alongside other analytical data to deduce composition of mixtures

R_f values can be calculated by taking 2 measurements from the TLC plate

• The less polar components travel further up the TLC plate
  • Their R_f values are higher than those closer to the baseline
  • They are more soluble in the mobile phase and get carried forwards with the solvent

• More polar components do not travel far up the plate
  • They are more attracted to the polar stationary phase

• The extent of separating molecules in the investigated sample depends on the solubility in the mobile and stationary phases
• Knowing the R_f values, of compounds being analysed, helps to compare the polarity of various molecules

Answers:

Blue spot R_f value = 2.82 ÷ 4.63=  0.61 = Leucine

Green spot R_f value = 2.45 ÷ 4.63= 0.536 = Isoleucine

Red spot R_f value = 2.04  ÷ 4.63= 0.44 = Valine