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Paper chromatography and Rf values

Set up paper chromatography, compare unknown and reference spots, calculate Rf and explain the limits of the evidence.

  • SEC G3 Pure Chemistry 2027

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A spot of ink can contain several substances. Paper chromatography separates small amounts of substances carried by a solvent, helping you compare a mixture with known reference samples.

Why the substances separate

The solvent rises through the paper. A dissolved substance moves with the solvent but can also be held back by attraction to the stationary material in the paper. Substances with different solubilities in the solvent and different attractions to the paper travel different distances under the same conditions.

A substance carried more readily by the solvent and held back less travels farther. It is the balance between these effects that matters; greater water solubility alone does not predict the result when a different solvent is used.

Set up and develop the chromatogram

Paper chromatography setupPaper hangs in a covered beaker. The pencil start line is above the solvent level. Three separated spots lie above one starting position and below the marked solvent front.solvent frontpencil start linesolventseparated spotscovered container reduces solvent evaporation
Paper chromatography: use a pencil start line above the solvent level and mark the solvent front before it evaporates.
  1. Draw a start line in pencil near the bottom of the paper. Add a small sample spot to the line and let it dry. Put reference samples in separate labelled lanes if you want to compare identities.
  2. Place the paper in a suitable solvent with its surface below the start line. A submerged sample can dissolve into the reservoir instead of travelling up the paper.
  3. Cover the vessel to reduce solvent evaporation. Allow the solvent to rise without letting it reach the top of the paper.
  4. Remove the paper and mark the solvent front immediately in pencil, before the solvent evaporates. Let the paper dry.

Pencil graphite does not dissolve and travel with the solvent. An ink start line could add extra spots, making the sample harder to interpret.

Make colourless spots visible

If the separated compounds are colourless, a suitable locating agent makes the spots visible after the chromatogram has developed. Otherwise, “I cannot see a spot” may mean that the method has not revealed it, rather than that the substance is absent. Knowledge of particular locating agents is not required for this course.

Compare an unknown with references

Compare an unknown with two reference samplesThree lanes on the same paper share a pencil baseline and solvent front. The unknown lane has two spots: an upper spot at the same height as reference A's single spot, and a lower spot at the same height as reference B's single spot. Both spots are above the baseline and below the solvent front. All lanes were run in the same solvent under the same conditions.unknownABsample lanessolventfrontpencilbaseline
An illustrative chromatogram from one solvent and one run. Compare each unknown spot with the reference spots at the same height. Matching positions support consistency with a reference; they do not prove identity in every solvent.

The unknown has two separated spots, so it contains at least two detected components. Its upper spot matches reference A’s position and its lower spot matches B’s. These matches support the presence of A and B, provided the samples were run under the same conditions; they do not prove the identities by themselves.

For valid comparisons, use the same solvent, paper and experimental conditions. A known substance’s position or R_f can change when the conditions change. One visible spot does not prove purity: different substances may travel together, and a detection method may not reveal every component.

Calculate the retention factor, Rf

Measure both distances from the same start line, along the solvent’s direction of travel. Measure to the centre of the spot, rather than its nearest or farthest edge.

R_f = (distance from start line to centre of spot)/(distance from start line to solvent front)

Measure the two distances for a retention factorAn illustrative paper chromatogram has a pencil start line at the bottom, a spot centre 3.0 centimetres above it and a solvent front 10.0 centimetres above it. Two vertical double-headed arrows share the start-line level and end at the spot centre and solvent front respectively.solvent frontspot centrepencil start line3.0 cm10.0cm
Measure both distances from the same pencil start line. The spot centre is 3.0 cm above it; the solvent front is 10.0 cm above it. The spot distance is the numerator, so Rf = 3.0/10.0 = 0.30.

In the diagram, the spot centre is 3.0 cm from the start line and the solvent front is 10.0 cm from it:

R_f = (3.0 cm)/(10.0 cm) = 0.30

The units cancel, so R_f has no unit. In an ordinary valid chromatogram the spot stays between the start line and solvent front, so 0 ≤ R_f ≤ 1. A value above 1 suggests an inverted ratio or incorrectly measured distances.

Run the chromatograms yourself: put the ruler’s zero on the start line, record a spot centre and the solvent front, and match the unknown with the known samples.

t = 0 min

Paper chromatography of food colouring X, red dye, blue dye, yellow dye in water, with the start line in pencil and the solvent below the start line. The solvent has not reached the start line yet. The ruler's zero is not on the start line and its cursor reads 4.0 cm.

Ruler reading at the cursor
4.0 cm
Rf from your readings
—
Samples
Start line drawn in

Try this

0 of 4 done

  1. Run one chromatogram with an ink start line and one with the solvent above the start line. (not done yet)

  2. When the run ends, put the ruler beside a lane with its zero on the start line. Record the distances to a spot's centre and to the solvent front. (not done yet)

  3. Find a known sample with a spot at the same Rf as a spot of the unknown in lane 1. (not done yet)

  4. Run the amino acids, then spray the paper with the locating agent. (not done yet)

Your readings

#laned / cmRemove
No readings yet. Set up a measurement, then record it.

Worked examples

Guided practice 1

Calculating R_f

About 5 min

Problem

On a chromatogram, a dye spot moves 3.0 cm from the start line while the solvent front moves 10.0 cm. Calculate R_f.

Choose numerator, denominator and result

Correct ratio
Rf value

Hints

Hint 1: formula
Distance travelled by spot goes above distance travelled by solvent front.
Hint 2: units
Both distances use cm, so their units cancel.
View solution step by step

  1. Substitute the distances

    Method

    Divide the spot distance by the solvent-front distance.

    Reason

    This is the definition of retention factor.

    Working

    R_f = 3.0/10.0.

  2. Calculate and check

    Method

    State R_f = 0.30 without a unit.

    Reason

    The distance units cancel and a valid value lies between 0 and 1.

    Working

    R_f = 0.30.

Common misconception 2

Error Analysis in Chromatography

Find and correct the mistake

Learner setup

A learner draws the start line with blue ink and places the solvent above that line. Explain and correct both errors.

Protect the reference line and sample spots

Start-line material
Solvent level

View solution step by step

  1. Correct the baseline

    Method

    Draw the start line in pencil.

    Reason

    Blue ink dissolves and separates, adding unwanted spots that confuse the chromatogram; graphite is insoluble.

    Working

    Use a pencil reference line.

  2. Correct the solvent level

    Method

    Keep the solvent below the start line.

    Reason

    If submerged, the samples dissolve into the reservoir instead of moving up the paper with the solvent front.

    Working

    Start line and sample spots above the solvent surface.

Try it independently

Mind stretcher 1: Compare within one runExtension

On the same chromatogram, the solvent front moves 8.0 cm. An unknown spot and reference C each move 4.8 cm. Calculate their R_f values and explain what the match supports. Does it prove that the unknown sample is pure C?

Show answer

Each R_f = 4.8/8.0 = 0.60, with no unit. The match is consistent with that unknown component being C under these conditions. It does not prove identity or purity: another substance could have the same R_f, and other components might not be detected or separated in this solvent.

Mind stretcher 2: Chromatography “Purity” TrapExtension

Question: A student runs a sample and sees one spot. The student concludes “the sample is pure”. Correct this conclusion.

Show Answer

One spot means one visible component under those conditions (same solvent, same paper). Different substances can sometimes have the same R_f in one solvent, so one spot does not guarantee purity.

Practise and check

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