Core Practical: Investigating Paper Chromatography

This lesson covers the Core Practical on paper chromatography as required by the Edexcel GCSE Combined Science specification (1SC0). You need to know how to carry out the practical, calculate Rf values, interpret chromatograms, and identify whether substances are pure or mixtures.

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Aim of the Practical

To use paper chromatography to separate and identify the coloured components (dyes) in food colourings or inks, and to calculate Rf values.

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Apparatus and Materials

Item	Purpose
Chromatography paper	The stationary phase
Pencil	To draw the baseline (insoluble in solvent)
Ruler	To measure distances
Beaker or chromatography tank	To hold the solvent
Solvent (e.g. water or ethanol)	The mobile phase
Capillary tube or spotting tile	To apply small spots of sample
Known reference dyes	To compare with unknown samples
Watch glass or cling film	To cover the beaker and prevent evaporation

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Method

Step-by-Step Procedure

1. Draw a baseline in pencil approximately 1–2 cm from the bottom of the chromatography paper.
2. Label the baseline with pencil marks to show where each sample will be placed.
3. Use a capillary tube to place a small spot of each sample on the baseline. Keep the spots small and concentrated — apply multiple times, allowing to dry between applications.
4. Pour the solvent (e.g. water) into the beaker to a depth of about 1 cm — the solvent must be below the baseline.
5. Lower the paper into the beaker so the bottom edge dips into the solvent, but the spots remain above the solvent level.
6. Cover the beaker with a watch glass or lid to prevent evaporation of the solvent.
7. Wait for the solvent to rise up the paper. Remove the paper when the solvent is about 1–2 cm from the top.
8. Immediately mark the solvent front with a pencil before it dries.
9. Allow the chromatogram to dry, then circle any spots visible.

graph TD
    A["1. Draw pencil<br/>baseline"] --> B["2. Spot samples<br/>on baseline"]
    B --> C["3. Place paper<br/>in solvent<br/>(below baseline)"]
    C --> D["4. Cover beaker<br/>and wait"]
    D --> E["5. Solvent rises<br/>by capillary action"]
    E --> F["6. Remove paper<br/>when solvent near top"]
    F --> G["7. Mark solvent<br/>front immediately"]
    G --> H["8. Dry and<br/>analyse spots"]

    style A fill:#2980b9,color:#fff
    style B fill:#2980b9,color:#fff
    style C fill:#27ae60,color:#fff
    style D fill:#27ae60,color:#fff
    style E fill:#e67e22,color:#fff
    style F fill:#e67e22,color:#fff
    style G fill:#c0392b,color:#fff
    style H fill:#c0392b,color:#fff

Exam Tip: Key details examiners look for: (1) baseline drawn in pencil, (2) solvent level below the baseline, (3) beaker is covered to prevent evaporation.

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Calculating Rf Values

The Rf value (retention factor) is a ratio that describes how far a substance travels relative to the solvent front. It is calculated using the formula:

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

Both distances are measured from the baseline (origin).

Example Calculation

Measurement	Value
Distance moved by solvent front from baseline	10.0 cm
Distance moved by substance A from baseline	7.5 cm
Distance moved by substance B from baseline	4.0 cm

• Rf of substance A = 7.5 ÷ 10.0 = 0.75
• Rf of substance B = 4.0 ÷ 10.0 = 0.40

Key Rules for Rf Values

• Rf values are always between 0 and 1 (the substance cannot travel further than the solvent).
• Rf values have no units.
• Rf values are specific to a particular substance and solvent combination.
• If the same solvent is used, an Rf value can be used to identify a substance by comparing it with known reference values.

Exam Tip: Rf values are only valid for comparison if the same solvent and same conditions (temperature, paper type) are used. Always measure from the centre of each spot to the baseline.

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Interpreting Chromatograms

Number of Spots

Observation	Conclusion
One spot	The substance is likely pure (a single component)
Two or more spots	The substance is a mixture containing multiple components

Matching Spots

• If a spot from an unknown sample is at the same height (same Rf value) as a known reference substance, the unknown likely contains that substance.
• If all spots in a mixture match known reference substances, the components of the mixture have been identified.

Example Chromatogram Analysis

Suppose you test three food colourings (X, Y, Z) alongside known reference dyes (Red, Blue, Yellow):

Sample	Spots Observed	Matches	Conclusion
X	1 spot	Matches Red reference	X contains red dye only (pure)
Y	2 spots	One matches Red, one matches Blue	Y is a mixture of red and blue dyes
Z	3 spots	Matches Red, Blue and Yellow	Z is a mixture of three dyes

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Common Exam Questions

Why Is the Baseline Drawn in Pencil?

Pencil (graphite) is insoluble in the solvent. If pen ink were used, the dye would dissolve and travel up the paper, contaminating the results.

Why Must the Solvent Level Be Below the Baseline?

If the solvent is above the baseline, the spots would dissolve directly into the solvent in the beaker rather than being carried up the paper. No separation would occur.

Why Is the Beaker Covered?

To prevent the solvent from evaporating, which would stop the solvent from rising up the paper and give unreliable results.

What If a Substance Is Colourless?

If the substances are colourless, you may need to use a locating agent (e.g. ninhydrin spray for amino acids) or view the paper under UV light to make the spots visible.

Exam Tip: When describing the method, use precise scientific language. Say "the solvent rises by capillary action" rather than "the water goes up the paper."

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Sources of Error and Improving Accuracy

Source of Error	Improvement
Spots are too large and overlap	Apply smaller, more concentrated spots
Solvent evaporates	Cover the beaker with a lid
Baseline drawn in pen	Use pencil (insoluble)
Solvent above baseline	Ensure solvent level is below the pencil line
Measuring from wrong point	Measure from the centre of each spot to the baseline
Not marking solvent front	Mark the solvent front immediately after removing the paper

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Summary

• Paper chromatography separates dissolved substances based on their solubility in the mobile phase.
• The Rf value = distance moved by substance ÷ distance moved by solvent front (measured from the baseline).
• Rf values are always between 0 and 1, have no units, and are specific to a particular solvent and conditions.
• A single spot indicates a pure substance; multiple spots indicate a mixture.
• Substances can be identified by comparing their Rf values with known reference substances under the same conditions.
• Key practical details: pencil baseline, solvent below baseline, covered beaker, mark solvent front immediately.

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Worked Example — Calculating Rf Values Step by Step

A student carries out paper chromatography on three food colourings (P, Q, R) alongside three known dyes (Red, Blue, Yellow). After the experiment, they measure:

Measurement	Distance from baseline (cm)
Solvent front	8.0
Red reference spot	4.0
Blue reference spot	2.0
Yellow reference spot	6.0
Dye P spot	6.0
Dye Q spot 1	4.0
Dye Q spot 2	2.0
Dye R spot	3.2