Chromatography and Separation

Chromatography is a technique used to separate and identify the components of a mixture. In A-Level Biology, you need to understand how chromatography works, how to calculate and interpret Rf values, and how related techniques such as electrophoresis can be used to separate biological molecules including amino acids, proteins, and nucleic acids.

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Principles of Chromatography

All chromatographic techniques rely on the differential partitioning of substances between two phases:

• Stationary phase: a fixed material that does not move (e.g., chromatography paper, thin-layer coating, gel in a column).
• Mobile phase: a liquid or gas that moves through or over the stationary phase, carrying the sample components with it (e.g., a solvent).

Different components of a mixture have different affinities for the stationary and mobile phases. Components that are more soluble in the mobile phase travel further and faster. Components that have a greater affinity for the stationary phase travel more slowly and remain closer to the origin.

Key Definition: Chromatography is a technique for separating the components of a mixture based on their differential solubility in (or affinity for) a stationary phase and a mobile phase.

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Paper Chromatography

Paper chromatography uses chromatography paper as the stationary phase and a solvent (e.g., a mixture of water and an organic solvent such as propanone or butanol) as the mobile phase. Water trapped in the fibres of the paper also acts as part of the stationary phase.

Method

1. Draw a pencil line (the origin line) near the bottom of a sheet of chromatography paper. Use pencil, not ink, because ink would dissolve in the solvent and interfere with the results.
2. Using a capillary tube or micropipette, place a small, concentrated spot of the sample on the origin line. If separating multiple samples or known standards, space them evenly along the line.
3. Allow the spots to dry completely. Apply more sample to the same spot if needed (to increase concentration and improve visibility).
4. Place the paper in a chromatography tank containing a shallow layer of solvent. The solvent level must be below the origin line so that the spots are not submerged.
5. Cover the tank with a lid to create a saturated atmosphere of solvent vapour (this prevents uneven evaporation from the paper).
6. The solvent moves up the paper by capillary action, carrying the dissolved components with it.
7. When the solvent front has nearly reached the top of the paper, remove the paper and immediately mark the position of the solvent front with a pencil line.
8. Allow the paper to dry in a fume cupboard.
9. If the separated components are colourless (e.g., amino acids), the paper must be treated with a locating agent such as ninhydrin (which produces purple/blue spots with amino acids) to make them visible.

Calculating Rf Values

Key Definition: The Rf value (retention factor or retardation factor) is the ratio of the distance travelled by the solute to the distance travelled by the solvent front, measured from the origin line.

Rf = distance moved by the solute from the origin / distance moved by the solvent front from the origin

Important points about Rf values:

• Rf values are always between 0 and 1 (a component cannot travel further than the solvent front).
• Each substance has a characteristic Rf value for a given solvent system, temperature, and type of paper. Rf values allow identification of unknown substances by comparison with known standards.
• Rf values are dimensionless (no units).
• Always measure distances from the centre of the spot to the origin line.

Worked Example:

A particular amino acid spot has moved 5.6 cm from the origin line. The solvent front has moved 8.0 cm from the origin line.

Rf = 5.6 / 8.0 = 0.70

If a table of known Rf values shows that leucine has an Rf of 0.70 under the same conditions, the amino acid is identified as leucine.

Two-Way Chromatography

If two or more components have very similar Rf values in one solvent and cannot be separated clearly, two-way (two-dimensional) chromatography can be used:

1. Run the chromatography in the first solvent as normal.
2. Dry the paper, then rotate it 90°.
3. Run the chromatography again using a different solvent.
4. Components that co-migrated in the first solvent may now separate because they have different Rf values in the second solvent.

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Thin-Layer Chromatography (TLC)

TLC operates on the same principles as paper chromatography but uses a thin layer of adsorbent material (usually silica gel or alumina) coated on a glass, plastic, or aluminium plate as the stationary phase.

Advantages over Paper Chromatography