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.

By the end of this lesson you should be able to: explain the principle of partitioning between a stationary and a mobile phase; describe paper and thin-layer chromatography methods and account for the differences between them; calculate and interpret Rf values, and explain why they depend on the solvent system; explain the principle of two-dimensional chromatography; describe gel electrophoresis and account for how DNA, RNA, and proteins are separated; and evaluate the sources of error and the reliability of separation techniques.

The partition coefficient — the physical basis of Rf

At A-Level depth, the differential migration that produces separation is governed by each solute's partition coefficient: the ratio of its solubility in (or affinity for) the mobile phase to its solubility in the stationary phase. A solute that dissolves readily in the mobile phase and weakly in the stationary phase has a high partition coefficient, spends most of its time moving, and therefore travels a long way up the paper — giving a high Rf. A solute that binds strongly to the stationary phase has a low partition coefficient, spends most of its time held in place, and travels only a short distance — giving a low Rf. Because this ratio is fixed for a given solute–solvent–stationary-phase combination at a given temperature, the Rf is reproducible and can be used for identification. Crucially, Rf is a property of the whole system, not of the molecule alone: change the solvent and the partition coefficient changes, so the Rf changes too. This is precisely why reference Rf values must always be quoted for a stated solvent system.

Worked Example — deducing composition from a two-way chromatogram

A student separates a mixture of amino acids by two-dimensional chromatography, using solvent 1 for the first run and solvent 2 (after a 90° rotation) for the second, then sprays with ninhydrin. Four purple spots appear, plus one yellow spot. The measured distances (from the origin, in the second-solvent direction) and the solvent front at 10.0 cm are:

Spot	Colour with ninhydrin	Distance in solvent 2 / cm	Rf (solvent 2)
P	Purple	8.5	0.85
Q	Purple	6.0	0.60
R	Purple	4.2	0.42
S	Purple	2.6	0.26
T	Yellow	3.0	0.30

Interpretation:

1. Five distinct spots means at least five different amino acids are present — two-dimensional chromatography has resolved components that a single run might have overlapped.
2. Spots P–S are purple: ninhydrin gives a purple/blue colour with most amino acids because it reacts with the free α-amino group.
3. Spot T is yellow, not purple. This is the diagnostic signature of proline (and hydroxyproline), whose nitrogen is part of a ring (a secondary amine), so it reacts with ninhydrin differently and yields a yellow product rather than the usual purple. Recognising the yellow spot as proline is a classic A-Level discriminator.
4. Each amino acid is then identified by matching both its first-solvent and second-solvent Rf values against reference standards run under identical conditions — using two Rf coordinates (one per dimension) makes identification far more reliable than a single value.

Exam Tip: If a question shows a ninhydrin chromatogram with one anomalously coloured (yellow) spot, name proline explicitly and explain the reason (secondary amine / imino group), then state that identification requires comparison of Rf values with known standards. The reasoning from spot colour to identity, plus the caveat that standards must be run under identical conditions, secures the AO3 marks.

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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

• Faster: solvent moves through the thin layer more quickly.
• Better resolution: spots are sharper and more distinct, giving better separation.
• More reproducible: manufactured plates have uniform thickness, leading to more consistent Rf values.
• Compatible with a wider range of solvents.

The method is otherwise very similar to paper chromatography: spot the sample, develop in solvent, mark the solvent front, locate spots (using UV light for fluorescent plates, or by staining), and calculate Rf values.

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Electrophoresis

Electrophoresis separates molecules based on their charge and size (molecular mass) when an electric field is applied across a gel.

Gel Electrophoresis

The most common form used in biology is gel electrophoresis, which separates proteins or nucleic acids (DNA/RNA fragments).

Method

1. Prepare an agarose gel (for DNA/RNA) or a polyacrylamide gel (for proteins, known as PAGE) with wells at one end.
2. Load the samples into the wells using a micropipette.
3. Place the gel in a buffer solution (to maintain pH and provide ions to carry the current) inside an electrophoresis tank.
4. Connect the electrodes and apply a voltage across the gel.
5. DNA and RNA are negatively charged (due to phosphate groups) and migrate toward the positive electrode (anode). Smaller fragments move faster through the gel matrix and travel further.
6. Proteins may be positively or negatively charged depending on their amino acid composition and the pH of the buffer. In SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis), the detergent SDS denatures proteins and coats them with a uniform negative charge proportional to their mass, so separation is based on size alone.
7. After running, the gel is stained to visualise the bands:
   • DNA: ethidium bromide (viewed under UV light) or safer stains such as SYBR Safe.
   • Proteins: Coomassie Blue stain.

Interpreting Electrophoresis Results

• Bands closer to the wells represent larger molecules (they moved more slowly through the gel).
• Bands further from the wells represent smaller molecules.
• A DNA ladder (molecular weight marker) with fragments of known sizes is run alongside the samples to estimate the sizes of unknown fragments.
• The distance migrated is approximately proportional to the log of the molecular mass for linear DNA and SDS-denatured proteins.

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Separating Amino Acids

Amino acids can be separated using:

1. Paper chromatography or TLC (as described above) — separation is based on solubility differences between amino acids in the mobile and stationary phases.
2. Electrophoresis — at a given pH, amino acids carry different net charges depending on their ionisable R groups. In an electric field:
   • Positively charged amino acids (e.g., lysine at neutral pH) move toward the cathode (negative electrode).
   • Negatively charged amino acids (e.g., aspartate at neutral pH) move toward the anode (positive electrode).
   • Neutral amino acids remain near the origin.

Identifying Amino Acids

After separation, amino acids are typically visualised using:

• Ninhydrin spray: reacts with amino acids to produce purple/blue spots (except proline, which gives a yellow spot).
• UV light (on fluorescent TLC plates): amino acids appear as dark spots against a bright background.

Identification is then carried out by comparing Rf values (chromatography) or migration distances (electrophoresis) with those of known standards run under identical conditions.

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Practical Considerations and Sources of Error

When performing chromatography or electrophoresis:

• Temperature control: Rf values change with temperature, so experiments should be carried out at a constant temperature. Electrophoresis generates heat, which can distort bands — use buffer and appropriate voltage.
• Solvent purity: impurities in the solvent can alter Rf values.
• Sample concentration: if the spot is too dilute, it may not be visible after development. If too concentrated, spots may be elongated (streaking) or overlap.
• Saturation of the tank atmosphere: in chromatography, the tank should be sealed to prevent evaporation from the paper/plate.
• Loading volume: in electrophoresis, overloading a well can cause smearing and poor resolution.
• Staining time: overstaining or understaining can make bands difficult to interpret.

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Summary

• Chromatography separates substances based on differential partitioning between a stationary and a mobile phase.
• Paper chromatography and TLC are used to separate amino acids, sugars, and other small molecules; Rf values allow identification.
• Rf = distance moved by solute / distance moved by solvent front (always between 0 and 1).
• TLC offers better resolution, speed, and reproducibility than paper chromatography.
• Gel electrophoresis separates DNA/RNA (by size) and proteins (by size in SDS-PAGE, or by charge and size) in an electric field.
• Amino acids are identified after separation using ninhydrin staining and comparison with known standards.
• Careful control of temperature, solvent composition, sample concentration, and technique is essential for reliable results.

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A-Level Deep Dive

Spec mapping

This lesson is mapped to AQA 7402 Section 3.5.1 — Chromatographic separation as it relates to Required Practical 6 — paper / TLC chromatography of plant pigments (refer to the official AQA specification document for exact wording). It covers the principles of partition chromatography, paper and thin-layer methods, Rf calculation, two-dimensional chromatography, gel electrophoresis (as a complementary separation), and method evaluation. This lesson is the central anchor for AQA RP6.

Required Practical 6 — Paper / TLC Chromatography of Plant Pigments (full anchor)

The canonical AQA RP6 method separates the photosynthetic pigments of a leaf — chlorophyll a, chlorophyll b, the carotenoids (β-carotene), and the xanthophylls (lutein, etc.).

Method: