All subjects · Chemistry · WCH14 (Unit 4)

Chromatography and high-resolution mass spectrometry

Chromatography separates the components of a mixture, and high-resolution mass spectrometry identifies them by their accurate relative molecular mass. Together they are used in forensic science and drug testing.

How chromatography works (spec 15.21)

Every type has a stationary phase (which does not move) and a mobile phase (which moves through it). Components separate because they have different relative affinities: one that is more strongly attracted to (or adsorbed on) the stationary phase, or less soluble in the mobile phase, moves more slowly.

Paper and thin-layer chromatography (spec 15.22)

In TLC the stationary phase is a thin layer of silica or alumina on a plate; in paper chromatography it is the paper (with water held in it). The solvent is the mobile phase.

Rf = distance moved by the spot / distance moved by the solvent front

Rf is always less than 1 and, for given conditions, is characteristic of a compound. Colourless spots are shown with UV light or a locating agent.

HPLC and GC (spec 15.23)

High-performance liquid chromatography pumps a liquid mobile phase at high pressure through a column packed with a solid. Gas chromatography carries a vaporised sample in an inert gas (such as helium or nitrogen) through a column whose stationary phase is a liquid on a solid support. Each component is identified by its retention time: the time from injection to detection. Peak areas show relative amounts. Coupled to a mass spectrometer (GC-MS or HPLC-MS), each separated component is identified from its mass spectrum: used in forensics, environmental analysis and testing athletes for banned drugs.

Accurate Mr (spec 15.17)

A high-resolution mass spectrometer measures m/z to four decimal places. Using accurate isotopic masses (1H 1.0078, 12C 12.0000, 14N 14.0031, 16O 15.9949), molecules with the same integer Mr can be told apart: CO 27.9949, N2 28.0062 and C2H4 28.0312.

Worked example

A molecular ion has m/z 60.0573. Is the compound C3H8O or C2H4O2?

  1. C3H8O: 3(12.0000) + 8(1.0078) + 15.9949 = 60.0573
  2. C2H4O2: 2(12.0000) + 4(1.0078) + 2(15.9949) = 60.0210

Answer: C3H8O

Key idea: Separation depends on relative affinity for the stationary and mobile phases. Rf = spot distance / solvent front distance. GC and HPLC use retention times; with MS they identify each component. Accurate Mr to 4 d.p. distinguishes formulae with the same integer mass.

Check yourself

  1. A spot moves 3.2 cm and the solvent front 8.0 cm. Calculate Rf.
    Show answer

    0.40. Rf = 3.2 / 8.0 = 0.40.

  2. In thin-layer chromatography, what is the stationary phase?
    • The solvent
    • A thin layer of silica or alumina on a plate
    • The sample
    • The air around the plate
    Show answer

    A thin layer of silica or alumina on a plate. The solid coating stays still while the solvent moves up the plate.

  3. Why do two compounds have different Rf values on the same plate?
    • They have different colours
    • They were spotted at different heights
    • One is heavier
    • They have different relative affinities for the stationary and mobile phases
    Show answer

    They have different relative affinities for the stationary and mobile phases. A compound adsorbed more strongly or less soluble in the solvent moves less.

  4. In gas chromatography, what is used to identify a component?
    • Its retention time
    • Its Rf value
    • Its colour
    • Its peak height only
    Show answer

    Its retention time. Retention time is characteristic under fixed conditions.

  5. Calculate the accurate relative molecular mass of C3H8O to four decimal places. (1H 1.0078, 12C 12.0000, 16O 15.9949)
    Show answer

    60.0573. 3(12.0000) + 8(1.0078) + 15.9949 = 60.0573.

  6. A molecular ion peak is at m/z 28.0312. Which molecule is it? (1H 1.0078, 12C 12.0000, 14N 14.0031, 16O 15.9949)
    • C2H4
    • CO
    • N2
    • CH2N
    Show answer

    C2H4. C2H4 = 24.0000 + 4.0312 = 28.0312; CO = 27.9949; N2 = 28.0062.

  7. Why is gas chromatography often coupled with mass spectrometry in drug testing?
    • MS separates the mixture and GC identifies each part
    • GC measures the accurate Mr directly
    • GC separates the mixture and MS identifies each separated component
    • It avoids needing a mobile phase
    Show answer

    GC separates the mixture and MS identifies each separated component. Retention times alone may be shared by different compounds; the mass spectrum confirms identity.

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Last updated 7 November 2026. Spotted a mistake? Tell us.