Thin-layer chromatography (TLC) separates the substances in a mixture as a solvent carries them up a plate coated with silica or alumina. Each substance moves a different distance, so the mixture separates into spots. This guide follows the practical as you would run it in the lab: spotting and developing a plate, making colourless spots visible, and identifying what is in a painkiller tablet from Rf values.
Core Specification Link
This practical sits in topic 3.3.16 Chromatography and links to 3.3.13 Amino acids, proteins and DNA, where amino acids are separated by TLC and located with ninhydrin or UV light. For the theory of stationary and mobile phases, see the thin-layer chromatography notes.
Aim
To separate the components of a mixture by thin-layer chromatography, locate the spots, and identify each component by comparing its Rf value with pure samples (standards) run on the same plate.
Equipment List
- TLC plate (silica gel on aluminium or plastic, containing a fluorescent indicator)
- Beaker (250 cm³) with a watch glass to use as a lid
- Solvent (mobile phase), for example ethyl ethanoate
- The mixture to be tested and pure samples of the suspected components, each dissolved in a little ethanol
- Glass capillary tubes (one for each sample)
- Pencil and ruler
- Tweezers
- UV lamp and UV-protective eyewear
- Locating agent if needed: iodine crystals in a sealed jar, or ninhydrin spray for amino acids
- Fume cupboard
Apparatus
The diagram shows a plate part of the way through developing. The solvent started below the baseline and has soaked up the plate, carrying each substance a different distance.
Method
- Hold the plate by its edges (or wear gloves). Draw a faint pencil baseline about 1 cm from the bottom, and mark a small cross on it for each sample, spaced well apart.
- Pour solvent into the beaker to a depth of about 0.5 cm, so it will be below the baseline. Cover the beaker with the watch glass and leave it for a few minutes so the air inside becomes saturated with solvent vapour.
- Spot each sample onto its own cross with a clean capillary tube, keeping each spot small (2 to 3 mm across). Let it dry, then add a little more to the same spot to concentrate it.
- Stand the plate in the beaker using tweezers, with the baseline above the solvent and the plate not touching the sides. Replace the lid straight away and do not move the beaker.
- Remove the plate when the solvent is about 1 cm from the top, and immediately mark the solvent front with a pencil line before the solvent evaporates.
- Let the plate dry in a fume cupboard, then locate the spots: view it under a UV lamp and draw round each dark spot in pencil, or use iodine vapour or ninhydrin.
- Measure the distances from the baseline to the centre of each spot, and from the baseline to the solvent front. Calculate each Rf value.
Why Each Step Matters
| Step | Reason |
|---|---|
| Baseline drawn in pencil | Graphite does not dissolve in the solvent, so the line stays in place. Ink contains dyes that would dissolve and separate into extra spots. |
| Solvent below the baseline | If the spots touched the solvent in the beaker, they would dissolve into it instead of being carried up the plate. |
| Lid on the beaker | The air stays saturated with solvent vapour, so solvent does not evaporate from the plate and the solvent front rises evenly. |
| Small, concentrated spots | Large spots spread out and overlap, so the components do not separate clearly and the distances are hard to measure. |
| Plate held by its edges | Fingers leave grease and amino acids from sweat on the plate, which can show up as extra spots, especially with ninhydrin. |
| Solvent front marked at once | The solvent evaporates quickly, and the position of its front is needed to calculate every Rf value. |
| Standards on the same plate | Rf values change with the solvent, the plate and the temperature. Running pure samples alongside the mixture keeps these conditions identical. |
Locating Colourless Spots
- UV light: the fluorescent indicator in the plate glows under a UV lamp. Compounds that absorb UV light, such as aspirin, paracetamol and caffeine, block the glow and appear as dark spots. Draw round them in pencil, because they cannot be seen once the lamp is off.
- Iodine vapour: put the dry plate in a sealed jar with a few iodine crystals. Many organic compounds show up as brown spots. Mark them straight away, because the colour fades.
- Ninhydrin (amino acids): spray the plate in a fume cupboard and warm it gently. Amino acids appear as purple spots. This is how the amino acids from a hydrolysed protein are identified.
Calculating Rf Values
\[ R_f = \frac{\text{distance moved by the spot}}{\text{distance moved by the solvent front}} \]Measure both distances from the baseline, going to the centre of each spot. Rf has no units and is always less than 1. A spot in the mixture with the same Rf value as a standard on the same plate is probably the same substance. To be more certain, run a second plate in a different solvent and check that they still match.
- A (aspirin): 5.4 cm
- P (paracetamol): 3.9 cm
- C (caffeine): 1.5 cm
- T (tablet): two spots, at 3.9 cm and 1.5 cm
Step 1: Calculate the Rf values of the standards
- Aspirin: 5.4 ÷ 7.5 = 0.72
- Paracetamol: 3.9 ÷ 7.5 = 0.52
- Caffeine: 1.5 ÷ 7.5 = 0.20
Step 2: Calculate the Rf values of the tablet's spots
3.9 ÷ 7.5 = 0.52 and 1.5 ÷ 7.5 = 0.20.
Step 3: Identify the components
The tablet's spots match paracetamol (0.52) and caffeine (0.20), which ran on the same plate under the same conditions. There is no spot at 0.72, so the tablet contains paracetamol and caffeine, but no aspirin.
Safety & Risk Assessment
| Hazard | Risk | Precaution |
|---|---|---|
| Ethyl ethanoate and other organic solvents | Highly flammable; the vapour can irritate the eyes and cause drowsiness. | Keep away from naked flames. Keep the beaker covered and work in a fume cupboard or well-ventilated room. |
| UV lamp | UV radiation can damage the eyes and skin. | Never look directly at the lamp. Wear UV-protective eyewear and switch the lamp off when not in use. |
| Iodine crystals and vapour | Harmful if inhaled or in contact with skin; stains skin and clothing. | Keep the jar sealed and use it in a fume cupboard. Wear gloves. |
| Ninhydrin spray | Harmful if swallowed; irritates the eyes and skin, and stains skin purple. | Spray only in a fume cupboard. Wear gloves and eye protection. |
Sources of Error & Improvements
| Error Source | Classification | Consequence & Mitigation |
|---|---|---|
| Spots too large or too concentrated | Random | The spots streak and overlap, so components with similar Rf values are not separated. Mitigation: Apply small spots, letting each application dry before adding more. |
| Judging the centre of a spot | Random | Spots are not perfectly round, so the distance to the centre is uncertain. Mitigation: Keep spots small, and let the solvent run close to the top so the distances are larger and the percentage uncertainty is smaller. |
| Solvent evaporating from the plate | Systematic | If the beaker is uncovered, the solvent front rises unevenly and the Rf values are unreliable. Mitigation: Keep the lid on, and saturate the beaker with vapour before adding the plate. |
| Two substances with the same Rf | Limitation | Different compounds can travel the same distance in one solvent. Mitigation: Run a second plate in a different solvent, or compare with more standards. |
Exam questions, marked instantly
Write your answer and the AI examiner checks it against the mark scheme, showing exactly which marks you earned and what to add.
Exam-style question
2 marksA spot from an unknown mixture has an Rf value of 0.45. Explain why a pure sample of the suspected compound should be run on the same plate, rather than comparing the Rf value with a data-book value.
Show Mark Scheme
- Rf values depend on the conditions, such as the solvent, the type of plate and the temperature [1]
- On the same plate the conditions are identical, so if the unknown spot travels the same distance as the pure sample, they are likely to be the same compound [1]
Exam-style question
3 marksA mixture is separated on a silica TLC plate using a non-polar solvent. Explain why the more polar component has the smaller Rf value.
Show Mark Scheme
- The silica stationary phase is polar [1]
- The more polar component is adsorbed more strongly onto the silica (stronger hydrogen bonds or dipole-dipole forces) and is less soluble in the non-polar solvent [1]
- So it spends more time on the stationary phase and moves a shorter distance up the plate [1]
CPAC Skills Assessed
- CPAC 1: Follows a written procedure to prepare, spot and develop a TLC plate.
- CPAC 3: Uses flammable solvents, UV light and locating agents safely.
- CPAC 4: Measures and records the distances needed to calculate Rf values.
Measure every distance from the pencil baseline: to the centre of each spot, and to the solvent front. When you identify a component, say that it has the same Rf value as a standard run on the same plate, not just the same value as in a data book.