Chromatography (Detailed)

Chromatography is a powerful separation technique used to identify and separate the components of a mixture based on differences in how strongly each component interacts with two competing phases: a stationary phase (which remains fixed) and a mobile phase (which moves through or over it). The word comes from the Greek 'chroma' (colour) and 'graphein' (to write), reflecting its early use in separating plant pigments. Today it is one of the most widely used analytical tools in chemistry, biology, forensic science, and medicine. In paper chromatography (PC), the stationary phase is the cellulose fibres of the paper (which hold a thin layer of adsorbed water), and the mobile phase is a liquid solvent that travels up the paper by capillary action. In thin-layer chromatography (TLC), the stationary phase is a fine powder — typically silica gel (SiO₂·xH₂O) or alumina (Al₂O₃) — coated onto a glass, aluminium, or plastic plate, and the mobile phase is again a solvent system. TLC gives sharper, faster separations than paper chromatography because the adsorbent particles are more uniform. Both techniques rely on the same principle: each component distributes itself between the stationary and mobile phases according to its relative affinity for each. A component with strong affinity for the mobile phase travels further up the plate or paper; one with strong affinity for the stationary phase barely moves. This differential migration produces separated spots on the chromatogram, which can then be identified using the retention factor (Rf value) or by comparison with known standards.

Key takeaways
  • Preparing the Baseline
  • Spotting the Sample
  • Development (Running the Chromatogram)
  • Stopping and Marking the Solvent Front
  • Locating the Spots
  • Interpreting the Chromatogram

Preparing the Baseline

A faint pencil line (never ink, which would itself separate) is drawn across the TLC plate or paper about 1–2 cm from the bottom. This is the baseline or origin. Pencil is used because graphite does not dissolve in solvents and will not migrate.

Spotting the Sample

A small, concentrated spot of the mixture is applied to the baseline using a capillary tube or micropipette. The spot must be small (ideally < 2 mm diameter) to ensure good resolution. If the sample is too dilute, the spot can be re-applied after the solvent has evaporated. Known reference compounds are spotted alongside for comparison.

Development (Running the Chromatogram)

The plate or paper is placed upright in a developing tank containing a small volume of solvent (the mobile phase). Crucially, the solvent level must be below the baseline so the spots are not submerged. The tank is covered to ensure a solvent-saturated atmosphere, which prevents uneven evaporation. The solvent rises by capillary action, carrying components with it at different rates.

Stopping and Marking the Solvent Front

When the solvent front reaches approximately 1–2 cm from the top of the plate, the plate is removed and the solvent front is immediately marked with a pencil before the solvent evaporates. This mark is essential for calculating Rf values.

Locating the Spots

Coloured compounds (such as plant pigments or ink dyes) are visible directly. Colourless compounds require locating agents or visualisation techniques: (a) UV light — many organic compounds fluoresce or quench fluorescence on UV-active TLC plates (containing a fluorescent indicator); (b) iodine vapour — iodine sublimes in a closed tank and reversibly stains most organic compounds brown/yellow due to the formation of weak charge-transfer complexes; (c) ninhydrin spray — reacts with amino acids to give purple/pink spots (Ruhemann's purple, formed by the reaction of ninhydrin with primary amines); (d) potassium manganate(VII) (permanganate) spray — oxidises organic compounds, producing yellow spots on a purple background; (e) 2,4-dinitrophenylhydrazine (2,4-DNPH) — detects carbonyl compounds (aldehydes and ketones), forming orange/red precipitate spots.

Interpreting the Chromatogram

Each spot represents one component. The number of spots in a mixture lane reveals the minimum number of components. A spot in the mixture lane that aligns with a spot from a known reference compound (same Rf value under identical conditions) suggests — but does not confirm alone — that the two substances are identical. For definitive identification, two-way chromatography (running in two perpendicular directions with different solvents) or combination with spectroscopic techniques (e.g. mass spectrometry, IR) is needed.

The Key Equation

Rf=distance travelled by spotdistance travelled by solvent frontR_f = \dfrac{\text{distance travelled by spot}}{\text{distance travelled by solvent front}}
  • R_f — Retention factor (retardation factor) (dimensionless (no units)): A number between 0 and 1 that characterises a compound's mobility under specific chromatographic conditions. It is constant for a given compound with a given stationary phase, mobile phase, and temperature.
  • d_{\text{spot}} — Distance travelled by spot (cm or mm): Measured from the baseline to the centre of the spot after development.
  • d_{\text{front}} — Distance travelled by solvent front (cm or mm): Measured from the baseline to the pencil mark indicating the solvent front position.

Worked Example

Problem. A TLC experiment is run to analyse a painkiller tablet. The solvent front travels 8.4 cm from the baseline. Three spots are observed in the tablet lane at distances of 1.2 cm, 3.6 cm, and 6.3 cm. A reference sample of aspirin (acetylsalicylic acid) produces a single spot at 3.6 cm. (a) Calculate the Rf value of each spot in the tablet lane. (b) Which component might be aspirin? (c) What does the presence of three spots indicate about the tablet?

Rf1=0.14,Rf2=0.43,Rf3=0.75R_{f1} = 0.14,\quad R_{f2} = 0.43,\quad R_{f3} = 0.75

Answer. Rf values: spot 1 = 0.14, spot 2 = 0.43, spot 3 = 0.75. Spot 2 is consistent with aspirin (Rf = 0.43). The three spots indicate at least three components in the tablet.

Figure
Diagram
TLC Chromatogram — Rf Value MeasurementMixtureReference AScalebaselinebaselinesolventfrontsame Rfdfront=8.4cmdspot=3.6cm● Spot 1 (Rf 0.14)● Spot 2 (Rf 0.43) = Ref A● Spot 3 (Rf 0.75)
Origins · 1 minMikhail Tsvet and how this was discovered
MT
Mikhail Tsvet
1872–1919 · Russian-Italian

Mikhail Tsvet invented chromatography around 1900 while studying plant pigments. He packed a glass column with calcium carbonate powder and poured a petroleum ether extract of chlorophyll through it, observing that different coloured pigments (chlorophylls and carotenoids) separated into distinct coloured bands — hence the name 'chromatography'. He published his landmark paper in 1906. His work was initially ignored but was rediscovered in the 1930s when column chromatography became widely used. Tsvet's surname (Цвет) means 'colour' in Russian — a remarkable coincidence for the inventor of a technique named after colour. He died of illness during the Russian Civil War at only 47, never seeing his invention become a cornerstone of analytical chemistry.

  • Mixtures and Pure Substances — Chromatography is used to separate and identify components of mixtures; understanding the distinction between mixtures and pure substances is essential context.
  • Separation Techniques — Chromatography belongs to the broader family of separation techniques (alongside distillation, filtration, and crystallisation), all of which exploit physical property differences.
  • Amino Acids and Proteins — Paper chromatography with ninhydrin locating agent is a classic method for identifying amino acids in hydrolysed protein samples, linking to organic and biochemistry topics.
  • Intermolecular Forces — The differential migration of components in chromatography is governed by intermolecular forces (hydrogen bonding, van der Waals forces, dipole interactions) between solute molecules and each phase.