C28: Chromatography
Paper chromatography method, calculating and using Rf values, interpreting chromatograms, and two-way chromatography for separating mixtures.
Chromatography
What is Chromatography?
Chromatography is a separation technique used to separate the components of a mixture. It works because different substances have different solubilities in the same solvent, so they travel at different rates through the stationary phase.
Chromatography has two phases:
- Mobile phase — the solvent that moves through the paper (e.g. water or ethanol).
- Stationary phase — the material that does not move (e.g. chromatography paper).
Components that are more soluble in the mobile phase travel faster and further. Components that are more attracted to the stationary phase travel slower and stay closer to the origin.
Paper Chromatography Method
Follow these steps to carry out paper chromatography:
- Draw a pencil baseline near the bottom of the chromatography paper. Never use ink — it would dissolve and run.
- Place small spots of each sample on the baseline using a capillary tube.
- Place the paper in a beaker containing a small amount of solvent. The solvent level must be below the baseline.
- Cover the beaker with a lid to prevent evaporation and keep the atmosphere saturated with solvent vapour.
- Allow the solvent to rise up the paper by capillary action.
- When the solvent nears the top, remove the paper and mark the solvent front with a pencil.
- Allow the paper to dry. If spots are colourless, use a locating agent or UV light to make them visible.
The result is a chromatogram — a pattern of spots showing where each component has moved to on the paper.
Rf Values
Rf = distance moved by substance / distance moved by solvent
Rf = a / b (where a = distance from baseline to centre of spot, b = distance from baseline to solvent front)
The Rf value is always between 0 and 1. A substance with an Rf value close to 1 is very soluble in the mobile phase and travels almost as far as the solvent front. A substance with an Rf value close to 0 is barely soluble and stays near the baseline.
The Rf value of a substance is constant for a given solvent at a given temperature. This means you can compare Rf values to identify unknown substances by matching them against known reference values.
Changing the solvent changes the Rf values because the solubility of each component changes. If a substance does not move in one solvent, try a different solvent.
Calculating Rf Values
Worked Example 1: Single Spot
A chromatogram is produced using water as the solvent. The solvent front travels 10.0 cm from the baseline. A spot from an unknown substance is found 6.5 cm from the baseline.
Rf = distance moved by substance / distance moved by solvent
Rf = 6.5 / 10.0 = 0.65
Looking up Rf reference tables, an Rf value of 0.65 in water matches the amino acid phenylalanine.
Worked Example 2: Multiple Spots
A mixture is separated by chromatography using ethanol as the solvent. The solvent front travels 8.0 cm. Two spots are observed:
- Spot A: 5.6 cm from the baseline → Rf = 5.6 / 8.0 = 0.70
- Spot B: 2.4 cm from the baseline → Rf = 2.4 / 8.0 = 0.30
By comparing with reference values in ethanol, Spot A can be identified as the component with Rf = 0.70 and Spot B as the component with Rf = 0.30.
Worked Example 3: Identifying Components
Three food dyes (X, Y, Z) and an unknown mixture are run on the same chromatogram. The solvent front is 12.0 cm from the baseline.
- Dye X spot: 8.4 cm → Rf = 8.4 / 12.0 = 0.70
- Dye Y spot: 4.8 cm → Rf = 4.8 / 12.0 = 0.40
- Dye Z spot: 1.2 cm → Rf = 1.2 / 12.0 = 0.10
- Unknown mixture: three spots at 8.4 cm, 4.8 cm and 1.2 cm
The unknown mixture contains all three dyes (X, Y and Z) because the spots appear at the same positions and have the same Rf values.
Interpreting Chromatograms
When interpreting a chromatogram:
- A pure substance produces a single spot on the chromatogram.
- A mixture produces two or more spots, because the different components separate out.
- Two substances are likely the same compound if they produce spots at the same height (same Rf value) when run on the same chromatogram.
- If a spot from a known reference is at the same position as a spot from an unknown sample, the unknown likely contains that substance.
Always measure to the centre of the spot when calculating Rf values, not the top or bottom edge.
Two-Way Chromatography
Sometimes different substances have the same Rf value in one solvent, so they do not separate properly. Two-way chromatography (also called two-dimensional chromatography) solves this problem by using two different solvents at right angles to each other.
Method for two-way chromatography:
- Carry out chromatography in the first solvent as normal.
- Allow the paper to dry completely.
- Turn the paper 90 degrees so the baseline is now vertical on one side.
- Run chromatography again using a different solvent.
- Components that did not separate in the first solvent may now separate in the second solvent because they have different solubilities.
Worked Example: Two-Way Chromatography
A mixture of amino acids is run in solvent 1 (butanol). Two amino acids (A and B) both have an Rf value of 0.45, so they appear as a single overlapping spot.
The chromatogram is turned 90° and run in solvent 2 (glacial acetic acid). In this solvent:
- Amino acid A has Rf = 0.30
- Amino acid B has Rf = 0.60
Now the two amino acids separate clearly because they have different solubilities in the second solvent. The two-way chromatogram gives better resolution and identification.
Factors Affecting Chromatography
| Factor | Effect on Results |
|---|---|
| Type of solvent | Different solvents give different Rf values for the same substance. Choose a solvent that gives good separation. |
| Temperature | Rf values change with temperature because solubility changes. Always compare Rf values obtained at the same temperature. |
| Baseline position | Must be above the solvent level, otherwise the samples dissolve into the solvent directly rather than being carried up the paper. |
| Spot size | Spots should be small and concentrated. Large spots spread out and overlap, making results unclear. |
| Paper type | Different chromatography papers have different properties. Use the same type for comparison experiments. |
| Drying | The paper must be allowed to dry before marking spots or applying a locating agent. |
Chromatography vs Other Separation Methods
| Method | Separates | Principle | Can Identify Substances? |
|---|---|---|---|
| Paper chromatography | Dissolved coloured or colourless substances | Different solubilities | Yes — using Rf values |
| Distillation | Liquids with different boiling points | Different boiling points | Partially — by boiling point |
| Filtration | Insoluble solids from liquids | Particle size | No |
| Evaporation | Solute from solvent | Difference in volatility | No |
In the exam, you may be asked to calculate an Rf value from a diagram. Make sure you measure distances carefully and remember: Rf has no units because it is a ratio of two distances.
If asked to explain why two substances might not separate in one solvent but do separate in another, refer to their different solubilities in each solvent. A substance more soluble in the mobile phase travels further.
Practice Questions
1. In a chromatography experiment, the solvent front moves 15.0 cm. A substance travels 9.0 cm from the baseline. Calculate the Rf value.
Rf = distance moved by substance / distance moved by solvent = 9.0 / 15.0 = 0.60
2. Explain why the baseline must be drawn in pencil, not ink.
Ink is soluble in the solvent and would dissolve and travel up the paper, producing its own spots and contaminating the results. Pencil graphite is insoluble and stays in place on the baseline.
3. A food colouring produces two spots on a chromatogram. Is the food colouring a pure substance? Explain your answer.
No, it is not a pure substance. A pure substance produces only one spot on a chromatogram. Two spots indicate that the food colouring is a mixture of at least two different components.
4. Describe when two-way chromatography is needed and how it works.
Two-way chromatography is needed when different substances in a mixture have the same Rf value in one solvent, so they do not separate. The chromatogram is turned 90° and run in a second, different solvent. Because the substances have different solubilities in the second solvent, they now separate and can be identified.
5. A student runs chromatography with the solvent level above the baseline. Explain what will happen and why.
The samples will dissolve directly into the solvent instead of being carried up the paper. The substances will wash off the paper into the solvent reservoir, and no chromatogram will be produced. The solvent level must be below the baseline so the solvent travels up through the samples by capillary action.
Required Practical
Paper Chromatography
Aim: To separate and identify the dyes in a mixture of food colourings using paper chromatography.
Method: Draw a pencil baseline about 1.5 cm from the bottom of the chromatography paper. Place small, concentrated spots of each known dye and the unknown mixture on the baseline using a capillary tube. Allow spots to dry. Pour the solvent (e.g. water or ethanol) into a beaker to a depth of about 1 cm. Suspend the paper so the bottom edge is in the solvent but the solvent level is below the baseline. Cover the beaker with a lid to prevent evaporation. Allow the solvent to rise up the paper until it is near the top. Remove the paper, mark the solvent front with pencil, and allow to dry.
Independent variable: The type of dye / substance being analysed
Dependent variable: Distance travelled by each component / Rf value
Control variables: Type of paper, solvent, temperature, baseline position, volume of solvent
Key points: Use pencil for the baseline (ink would run). Spots must be small and concentrated. Solvent level must be below the baseline. A lid prevents solvent evaporating and gives reproducible results.
Maths Skills
Calculating Rf Values
The Rf value (retardation factor) identifies substances in chromatography.
Formula: Rf = distance travelled by substance ÷ distance travelled by solvent
Example: A spot travels 4.2 cm from the baseline. The solvent front is 8.4 cm from the baseline.
Rf = 4.2 ÷ 8.4 = 0.50
Important points:
- Rf values are always between 0 and 1.
- Rf values have no units (it is a ratio).
- Measure from the baseline (where the spot was originally placed) to the centre of the spot, not the edge.
- The same substance has the same Rf value in the same solvent under the same conditions.
- Different solvents may give different Rf values for the same substance, so the solvent must always be stated.
Identification: Compare the Rf value of the unknown spot with Rf values of known substances run under the same conditions. If they match, the substances are likely the same.
Common Misconceptions
Misconceptions About Chromatography
The highest spot on the chromatogram travelled the furthest.
The spot with the highest Rf value travelled the furthest relative to the solvent front. A spot may appear high on the paper simply because the solvent front was very high. Always compare using Rf values (distance from baseline), not just visual position. The highest Rf value indicates the substance most soluble in the mobile phase solvent, not necessarily the "highest" spot visually.
Chromatography proves two substances are identical if their spots are at the same height.
The same Rf value in one solvent suggests the substances could be the same, but it does not prove it. Two different substances could coincidentally have the same Rf value in one solvent. Two-way chromatography (turning the paper 90° and using a different solvent) is used to confirm identity.
A pen should be used to draw the baseline on chromatography paper.
Only pencil should be used. Pen ink would dissolve in the solvent and travel up the paper, contaminating the results and producing extra spots.
6-Mark Question
Describe how to carry out paper chromatography and calculate Rf values.
Draw a pencil line about 1.5 cm from the bottom of a piece of chromatography paper. Place concentrated spots of the known reference substances and the unknown mixture on the baseline using a capillary tube, allowing each spot to dry before adding another drop for concentration. Allow all spots to dry completely. Pour a small amount of solvent into a beaker to a depth of about 1 cm. Carefully lower the paper into the beaker so the bottom edge is in the solvent, ensuring the solvent level is below the pencil baseline. Cover the beaker with a watch glass or lid to reduce evaporation. Allow the solvent to travel up the paper by capillary action until it is about 1 cm from the top. Remove the paper and immediately mark the solvent front with a pencil line. Allow the paper to dry. Measure the distance from the baseline to the centre of each spot, and measure the distance from the baseline to the solvent front. Calculate the Rf value for each spot: Rf = distance travelled by substance ÷ distance travelled by solvent. Compare the Rf values of the unknown spots with the Rf values of the known reference substances to identify the components.
AO3: Analysis and Evaluation
Evaluating Chromatography Results
A student ran paper chromatography on three food colourings (A, B and C) alongside four known dyes (1–4). The solvent front was 10.0 cm from the baseline. The results were:
- Dye 1: Rf = 0.20, Dye 2: Rf = 0.45, Dye 3: Rf = 0.60, Dye 4: Rf = 0.85
- Colouring A: one spot at Rf = 0.45
- Colouring B: two spots at Rf = 0.20 and Rf = 0.85
- Colouring C: two spots at Rf = 0.45 and Rf = 0.60
Evaluate which dyes each food colouring contains, and explain whether any colouring could be a pure substance.
Answer: Colouring A contains only Dye 2 (matching Rf = 0.45) and could be a single pure substance. Colouring B contains Dyes 1 and 4 (Rf = 0.20 and 0.85) — it is a mixture. Colouring C contains Dyes 2 and 3 (Rf = 0.45 and 0.60) — it is also a mixture. Only Colouring A is a pure substance since it produces a single spot. However, to confirm identity, the experiment should be repeated with a different solvent to check that the Rf values still match, as two different substances could have the same Rf value in one solvent by coincidence.
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