BTEC Applied Science – Unit 2, Learning Aim C
Chromatography is one of the most important analytical techniques in science. In Unit 2, Learning Aim C, you’ll explore how chromatographic methods such as paper chromatography and thin-layer chromatography (TLC) can separate and identify substances in a mixture.
These techniques are widely used in forensic science, food testing, and pharmaceutical analysis — so understanding how they work is essential for your assignments and practical assessments.
What Is Chromatography?
Chromatography works on a simple principle:
“Different substances move through a medium at different speeds.”
A mixture is dissolved in a mobile phase (a liquid or gas) and travels through a stationary phase (a solid or a liquid supported on a solid). Substances that interact strongly with the stationary phase move more slowly; those that interact weakly move further. This difference in movement allows the substances to be separated and identified.
Paper Chromatography
In paper chromatography, the stationary phase is the cellulose paper, and the mobile phase is a solvent such as water or ethanol. To carry out paper chromatography:
Draw a pencil line near the bottom of the paper.
Place small drops of your sample (e.g. inks or dyes) on the line.
Stand the paper in a beaker with a shallow layer of solvent — below the pencil line.
As the solvent moves up the paper, it carries the dyes with it.
The components separate because they have different solubilities in the solvent and different attractions to the paper fibres.
Once the solvent front has moved a suitable distance, the paper is removed and dried — this is called a chromatogram.
Calculating Rf Values
Each spot’s position can be measured using its Rf value:
Rf values are always less than 1, and they can be compared to known reference values to help identify substances.
Thin Layer Chromatography (TLC)
TLC works on the same principle as paper chromatography, but instead of paper, the stationary phase is a thin layer of silica gel or alumina spread on a glass or plastic plate.
The sample is spotted near the bottom of the TLC plate.
The plate is placed in a developing chamber containing the solvent.
As the solvent travels up, components separate based on their attraction to the stationary phase (silica or alumina) and their solubility in the mobile phase (the solvent).
Why Use TLC?
It gives sharper, faster separations than paper chromatography.
It can handle smaller sample sizes.
Plates can be visualised using UV light or chemical stains, revealing colourless compounds.
TLC is often used in drug purity testing, food additive analysis, and organic synthesis monitoring.
Factors Affecting Chromatography Results
Several factors influence how well the substances separate:
Factor | Effect | Explanation |
Solvent polarity | Determines how well the solutes dissolve and move | Polar solvents carry polar molecules further; non-polar solvents favour non-polar solutes |
Stationary phase | Controls adsorption strength | Silica (polar) attracts polar molecules; cellulose retains polar compounds |
Temperature | Affects solvent movement | Higher temperature can increase Rf values and reduce separation time |
Sample size | Affects clarity | Too much sample can cause streaking and poor separation |
Time in solvent | Affects distance moved | Longer time = greater travel, but excessive time may distort results |
Interpreting Results
When analysing your chromatogram:
Each spot represents a different compound.
Rf values can be compared to known standards.
The number of spots indicates how many substances were present in the original mixture.
If two samples have the same Rf value in the same solvent, they may be the same compound.
Remember: environmental conditions (temperature, humidity) can cause small differences, so results should always be compared under the same conditions.
Example Application
In a forensic investigation, chromatography might be used to match an ink sample from a ransom note to a suspect’s pen. In a food lab, it could identify artificial dyes in sweets. In pharmaceuticals, TLC checks whether a drug is pure or contains impurities.
Key Takeaways
Both paper and TLC separate mixtures using a stationary phase and a mobile phase.
Substances separate due to differences in solubility and adsorption.
The Rf value helps identify compounds.
TLC gives more precise, faster results and is widely used in modern labs.
Understanding these factors is essential for accurate analysis, identification, and evaluation in Unit 2 practicals.
Practice Question
“Explain how the choice of solvent affects the separation of amino acids in a chromatography experiment.”
✅ Model Answer (4 marks):The solvent acts as the mobile phase, carrying the amino acids up the stationary phase. A polar solvent interacts strongly with polar amino acids, causing them to move further up the plate. If the solvent is too non-polar, polar amino acids will move very little. Therefore, choosing a solvent of suitable polarity ensures good separation and clear results.
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