Paper Chromatography: Method and Rf Values

Put a dot of black felt-tip ink on a strip of filter paper, dip the end in water, and watch. Within minutes, the single black dot stretches into a streak of blue, purple, yellow and pink. The “black” ink was never one substance at all; it was a mixture of dyes, and paper chromatography has pulled them apart.

It’s one of the simplest and most striking experiments in chemistry, and the same principle underpins powerful techniques used in forensic science, drug testing and food analysis.

The principle

Every chromatography technique has two phases:

• The stationary phase doesn’t move. In paper chromatography, it’s the water held within the cellulose fibres of the paper.
• The mobile phase moves through the stationary phase. It’s the solvent (water, ethanol, or a mixture) that travels up the paper by capillary action.

As the solvent moves up the paper, it carries the substances in the mixture with it. Each substance spends part of its time dissolved in the moving solvent and part of its time held by the stationary phase. The balance between the two is different for each substance:

• A substance that’s very soluble in the solvent and weakly held by the paper spends more time in the mobile phase and travels further.
• A substance that’s less soluble or more strongly attracted to the paper travels less far.

This difference separates the mixture into distinct spots. For more on the general theory, see chromatography.

Equipment

• Chromatography paper (or good-quality filter paper)
• Pencil and ruler
• Capillary tubes or fine glass rods for spotting
• Samples: inks, food colourings, plant pigments, or solutions of known substances
• Beaker or chromatography tank with a lid (a watch glass or cling film works)
• Solvent (water for water-soluble dyes; ethanol, propanone or mixtures for others)
• Paper clip or splint to hang the paper
• Eye protection

Method

1. Draw a start line (baseline) in pencil about 1.5–2 cm from the bottom of the paper. Pencil is used because graphite is insoluble and won’t move; ink would itself separate and ruin the result.
2. Mark small crosses on the baseline, at least 1 cm apart, for each sample.
3. Spot each sample onto a cross using a capillary tube. Keep spots small (2–3 mm). Let each spot dry and re-spot two or three times to build up concentration without spreading the spot.
4. Label each spot in pencil below the line.
5. Add solvent to the beaker to a depth of about 1 cm, so the level is below the baseline. If the spots are under the solvent, they’ll dissolve into it instead of travelling up the paper.
6. Hang the paper so its bottom edge dips into the solvent without touching the sides of the container.
7. Cover the container with a lid. This keeps the air inside saturated with solvent vapour, so the solvent doesn’t evaporate from the paper and moves up evenly.
8. Leave the solvent to rise until it’s about 1–2 cm from the top.
9. Remove the paper and immediately mark the solvent front (the highest point the solvent reached) with a pencil line.
10. Let the chromatogram dry.

Calculating Rf values

The retention factor (Rf) compares how far a substance moved with how far the solvent moved:

Rf = distance moved by the substance ÷ distance moved by the solvent

Both distances are measured from the baseline. Measure to the centre of each spot.

Rf always lies between 0 and 1 and has no units.

Worked example:

• Solvent front: 8.0 cm from the baseline
• Spot A: centre 3.6 cm from the baseline
• Spot B: centre 6.4 cm from the baseline

Rf(A) = 3.6 ÷ 8.0 = 0.45 Rf(B) = 6.4 ÷ 8.0 = 0.80

B is more soluble in the solvent (or less attracted to the paper) than A.

More practice is in calculating Rf values.

Identifying substances

Under the same conditions (same paper, solvent and temperature), a particular substance always has the same Rf value. That lets you identify unknown components:

1. Spot the unknown mixture alongside reference samples of substances you suspect it contains.
2. Run the chromatogram.
3. If a spot in the mixture has the same Rf and the same colour as a reference spot, it’s probably the same substance.

Example: a food colouring gives three spots. Two match the Rf values of reference dyes E102 (tartrazine, yellow) and E133 (brilliant blue). The third matches none of the references, so it’s an unknown dye.

Pure or impure? A pure substance gives one spot (in any solvent). A mixture gives two or more. However, two different substances can occasionally have very similar Rf values in one solvent, so running a second solvent is good practice before concluding that a single spot means a pure sample.

What about colourless substances?

Many substances, such as amino acids and sugars, are colourless. After running and drying the chromatogram, they can be made visible with a locating agent:

• Ninhydrin spray reacts with amino acids to give purple or blue spots after gentle heating.
• Ultraviolet light shows spots of substances that fluoresce or absorb UV (usually with TLC plates containing a fluorescent indicator).
• Iodine vapour stains many organic compounds brown.

Locating agents are hazardous (ninhydrin is harmful and irritant), so they’re sprayed in a fume cupboard by a teacher or technician.

Two-way chromatography

When a mixture contains substances with similar Rf values, a single run may not separate them. In two-way chromatography:

1. Spot the mixture near one corner of a square sheet.
2. Run it in solvent 1.
3. Dry the paper, turn it 90°, and run it in solvent 2.

Substances that overlapped in the first solvent often separate in the second, giving a map of spots across the sheet. This technique was historically important in separating amino acids from proteins.

Sources of error and how to reduce them

Problem	Cause	Fix
Spots are streaky or smeared	too much sample; spots too large	smaller, more concentrated spots
Spots dissolve into the solvent	baseline below the solvent level	keep the solvent level below the baseline
Uneven solvent front	container not covered; paper touching the sides	use a lid; hang the paper freely
Rf values differ between groups	different solvent, paper or temperature	keep conditions identical and run references on the same sheet
Baseline smudges and moves	drawn in pen	always use pencil
Solvent front not marked	removed paper and forgot	mark it immediately

Real-world uses

• Forensics: comparing inks on forged documents, or dyes in fibres.
• Food science: checking food colourings against permitted additives.
• Biochemistry: separating amino acids, sugars and plant pigments.
• Education: the most accessible introduction to separation science.

Today, laboratories often use thin-layer chromatography (TLC), gas chromatography and HPLC, which are faster and more precise, but the principle is exactly the same. See thin-layer chromatography.

Safety

• Wear eye protection.
• Organic solvents such as ethanol and propanone are flammable: no naked flames nearby, and use in a well-ventilated area or fume cupboard.
• Locating agents such as ninhydrin should only be handled by staff, in a fume cupboard.

Key takeaways

• Paper chromatography separates mixtures by how substances distribute between a moving solvent and the stationary phase in the paper.
• Draw the baseline in pencil and keep the solvent level below it; cover the container.
• Rf = distance moved by substance ÷ distance moved by solvent, both from the baseline.
• Matching Rf values and colours with reference samples under the same conditions identifies substances.
• Colourless substances are revealed with locating agents such as ninhydrin or UV light.