Introduction to chromatography: TLC, GC, HPLC

Introduction to chromatography: TLC, GC, HPLC


In this lesson, we will learn:

  • How TLC is used in the laboratory as an analytical method.
  • The definitions of mobile phase, stationary phase and retention factor (Rf).
  • To calculate Rf values from given TLC experiment results.
  • The uses of HPLC and GC as types of column chromatography.


  • Chromatography is another analytical method that helps to identify individual compounds in a mixture of substances. You put in a mixture, and chromatography identifies and/or separates it into its different constituent compounds, normally due to their solubility.
    There are many types of chromatography but they all ultimately do this. There are two key parts to a chromatographic experiment:
    • The mobile phase, a liquid or gas (something that flows) that carries the analyte, the substance being analysed.
    • The stationary phase, a solid or fixed surface used for the mobile phase to move across. The stationary phase is usually very different in chemical structure to the mobile phase (such as nonpolar mobile phase and polar stationary phase).

    In short, chromatography separates mixtures because each compound has different interactions with the stationary and mobile phase; some interact more with polar and some with nonpolar. This leads to a different rate of flow, which leads to a different retention time or ‘position’ on the stationary phase when the analysis ends.

  • A simple and very common type of chromatography is thin layer chromatography. In this type of chromatography:
    • The stationary phase is a thin layer of silica on a glass plate. Added to this silica is a substance which responds to UV light. A small amount of the analyte mixture is ‘spotted’ on a horizontal pencil line on the plate near the bottom. This is drawn so that (if you have them) more than one mixture can be analysed at the same time.

    • The mobile phase is a solvent which dissolves the analyte mixture(s). It sits in the bottom of a beaker before the silica plate is added and the beaker is covered with a watch glass.

    • As the solvent travels up the silica plate (the stationary phase), the solvent reaches and dissolves the analyte mixture(s) and continues moving up the plate.

    • After a few minutes, the watch glass is removed and the TLC silica plate is removed from the solvent. A horizontal pencil line is drawn across the plate where the solvent reached – this is called the solvent front.

    • The TLC plate is then exposed to UV light. As said above, the plate reacts to the UV light; the analyte compounds do not. Any compounds in the analyte will stand out as dark spots against the UV-reactive TLC plate. These compound spots should be circled with a pencil. Because their movement on the TLC plate depends on their solvent solubility, each compound will move at its own ‘speed’ and have its own ‘spot’.

    • The ratio of the distance travelled by the compound compared to the distance travelled by the solvent is known as the retention factor (Rf) . It is calculated by:

    • Rf = DistancetravelledbyspotDistancetravelledbysolvent\large \frac{\text Distance \, travelled \, by \, spot}{\text Distance \, travelled \, by \, solvent}

      Rf is a just a distance (usually cm) divided by another distance so it is always unitless.

      The number of spots on the TLC plate tells you the number of different compounds in the analyte. Keeping the solvent the same, Rf can be used to identify particular compounds, for example in doing a repeat experiment or in comparing products of similar reactions.

  • Worked example: Calculating retention factor (Rf).
    A TLC experiment is run on the product mixture of “reaction 1” and two spots are found. Spot 1 (S1) is at 1.4 cm on the plate; spot 2 (S2) is at 3.5 cm. The solvent line is at 5.2 cm. Calculate the Rf values for S1 and S2.

  • A TLC experiment is run on the product mixture of “reaction 2”. The solvent used is the same in both TLC experiments. With the solvent line at 4.5 cm, spot 1 (S1) is at 0.9 cm on the plate; spot 2 (S2) at 1.5 cm and spot 3 (S3) is at 2.4 cm. Are any of the products of reaction 1 and reaction 2 potentially the same?

    If any of the spots are due to the same compound, their Rf values will match in both experiments, since the solvent is the same (and the time taken doesn’t matter since we divide by the solvent distance).
    First, we need to calculate the Rf values.
    • Experiment 1:
      • Rf of S1 = 1.4 cm / 5.2 cm = 0.27
      • Rf of S2 = 3.5 cm / 5.2 cm = 0.67
    • Experiment 2:
      • Rf of S1 = 0.9 cm / 4.5 cm = 0.2
      • Rf of S2 = 1.5 cm / 4.5 cm = 0.33
      • Rf of S3 = 2.4 cm / 4.5 cm = 0.53

    With the Rf values calculated, knowing that the solvent was the same, we expect a given compound to show the same Rf value across separate experiments. We can see this isn’t the case; we have 5 different Rf values across the two experiments. We can therefore say there are no products in common in either experiment.

  • TLC is a relatively quick and simple type of chromatography. There are more sophisticated column chromatography methods such as:
    • Gas chromatography: The stationary phase is a polymer coating on a long glass tubing called a column. The mobile phase is an inert gas that carries the vaporised analyte through the column.
      The carrier gas does not chemically interact with the analyte but the polymer stationary phase does. The stronger the interactions with the polymer, the longer it will take the analyte to ‘elute’ – pass through the column.
      This time taken to elute is the retention time (like Rf in TLC).
      GC is often used alongside mass spectrometry to find the molecular mass of individual compounds in the mixture.

    • High-performance liquid chromatography (HPLC): Instead of gas, the mobile phase is a pressurised liquid pumped through the column, but otherwise this is largely the same as GC. It will be used instead of GC when an analyte contains substances that decompose when heated.

    Like with Rf values in TLC, retention times with certain conditions in HPLC and GC can be used to match with reference values in chemical databases. This can identify particular compounds, so HPLC and GC are very useful in forensic science and drug-testing in professional sports.
  • Introduction
    Thin layer chromatography
    The basics of chromatography.

    How TLC (thin layer chromatography) works.

    Calculating the Rf value.

    GC and HPLC.

  • 1.
    Calculate the Rf values from TLC experimental results.
    A TLC experiment is run on the product mixture of a reaction and two spots are found. A compound at spot 1 (S1) is measured at 1.2 cm on the plate; another compound at spot 2 (S2) is measured at 3.0 cm. The solvent line is at 4.2 cm.
    1. Calculate the Rf values for S1 and S2.
    2. Explain why Rf values are always less than or equal to 1.