Unit 28: Chromatography — Long Questions

Principle Paper chromatography separates the components of a mixture based on their relative solubility (partition) between two phases — a mobile phase (the solvent moving up the paper) and a stationary phase (water held within the cellulose fibres of the paper). Components that are more soluble in the moving solvent travel further up the paper, while those more soluble in the water trapped in the paper travel less, so the mixture separates into distinct spots.

Working 1. A baseline is drawn in pencil near the bottom of a strip of chromatography paper.
2. A small spot of the sample is applied on the baseline using a capillary tube.
3. The paper is dipped into a suitable solvent, keeping the solvent level below the spot (otherwise the sample would dissolve straight into the solvent rather than travel up the paper).
4. The solvent rises up the paper by capillary action, carrying the dissolved components upward at different rates depending on their relative solubility in the two phases.
5. When the solvent has risen sufficiently, the paper is removed and the solvent front (the highest point reached by the solvent) is marked.
6. Coloured components are visible directly; colourless components are visualised by spraying with a suitable locating agent.
7. The Rf value of each spot is calculated: Rf = distance travelled by the substance ÷ distance travelled by the solvent. Rf has no units, as it is a ratio, and is constant for a given substance under the same solvent and temperature, so components can be identified by comparing their Rf values with standard values.
An advantage of this technique is that it needs only a very small quantity of the mixture.

Principle TLC separates the components of a mixture by a combination of adsorption onto a solid stationary phase and their relative solubility in a liquid mobile phase. Polar substances have a greater attraction for the polar solid stationary phase (silica or alumina) and are adsorbed onto it more strongly, so they move slowly; non-polar substances are more soluble in the (non-polar) solvent and move further/faster up the plate. This difference allows the components of the sample to separate.

Working 1. Alumina (Al2O3) or silica (SiO2) is mixed with water to make a slurry, spread evenly on a glass slide, and dried in an oven to form a solid white stationary layer on the slide.
2. A straight baseline is drawn in pencil, using a ruler, near the bottom of the slide.
3. A spot of sample is applied on the baseline with a capillary tube; more than one sample can be spotted at different places on the same slide.
4. The glass slide is placed in a chromatography tank and dipped in the solvent (mobile phase), with the baseline kept above the solvent level.
5. As the solvent rises, the components of the sample dissolve in it and adsorb onto the stationary phase to different extents — more strongly adsorbed (usually more polar) substances move slowly, while substances more soluble in the solvent move further.
6. Once the solvent has moved up to a suitable height, the slide is removed and the solvent front (the highest point reached by the solvent) is marked, and the slide is dried.
7. Rf values of the different spots are calculated (distance travelled by spot ÷ distance travelled by solvent) and compared with standard Rf values to identify the components. Colourless substances are visualised using a locating agent spray — for example, ninhydrin gives colourless amino acids a purple colour; some substances fluoresce under UV light, and others turn brown with iodine vapours.

Applications identifying unknown compounds by comparing Rf values, checking purity of substances, monitoring the progress of a reaction, checking purity of drugs and detecting preservatives/colourants/sweeteners in food, detecting pollutants/pesticides/toxins, and forensic identification of drugs, blood, sweat or semen at a crime scene.

Limitations TLC is mainly a qualitative technique and is not very accurate/precise for quantitative work; it cannot clearly separate compounds with close Rf values; only small amounts of sample can be applied, so it is unsuitable for large-scale separation; and it is sensitive to humidity, temperature and cleanliness of the working environment, which reduces reproducibility.

1) Carrier gas (mobile phase): an inert gas such as helium or nitrogen that carries the vaporised sample through the system.
2) Injector port: the point where the sample is injected; it is heated by an oven so the sample is rapidly vaporised before being carried into the column.
3) Column: a long, coiled column packed with tiny particles of silica (or alumina) coated with a non-volatile, long-chain, non-polar hydrocarbon liquid of high boiling point (the stationary phase). Separation occurs here: non-polar substances interact more strongly with the non-polar stationary phase and move through the column more slowly, while other substances move faster, so the mixture separates.
4) Detector: positioned where the separated substances exit the column; it identifies and records each component as a peak on a graph (the chromatogram) and also records its retention time (the time taken for the substance to pass through the column).
5) Chromatogram/recorder: the output trace of detector response versus time. Retention time is used to identify each component (by comparison with known values), and the area under each peak (or its height, when peaks are narrow/have similar base widths) is measured to give the proportion of each component in the mixture, using % of A = peak area of A ÷ sum of peak areas of all components × 100 (peak area ≈ ½ × base × height, since peaks are roughly triangular).