Part 1: TLC and Required Practical 12

Explain chromatographic separation, plan AQA Required Practical 12 and interpret TLC spots and Rf values with appropriate limitations.

Separation depends on two phases

Chromatography separates components because they distribute differently between a mobile phase that moves and a stationary phase that stays in place. A component that spends a greater proportion of time in the moving phase generally travels faster under the same conditions.

In thin-layer chromatography, TLC, the stationary phase is usually a thin layer of silica or alumina on a support and the mobile phase is a liquid solvent or solvent mixture. Attraction to the solid surface is adsorption. Absorption means entering the bulk of a material; do not use the two words as though they always describe the same process.

Explain separation as a balance between solubility or affinity in the mobile phase and attraction to or retention on the stationary phase. Saying only “different solubilities” leaves out half of the comparison. AQA June 2023 Paper 3 Q03.5 and its report explicitly address that omission.

On a polar silica stationary phase with a relatively non-polar mobile phase, a strongly adsorbed polar component may travel less far than a less strongly retained component. This is a conditional comparison, not a universal rule that every polar molecule always has a low Rf.

Required Practical 12: prepare a useful TLC separation

RP12 requires separation of species by TLC. The AQA handbook uses components of medicines as an example. A useful investigation runs the mixture alongside authentic reference substances under the same conditions. Prepare suitably dilute samples in a compatible solvent and use the centre’s supervised, risk-assessed procedure.

Draw a faint pencil baseline above the intended solvent level. Pencil graphite stays in place; ink may dissolve and separate, adding misleading spots. Apply small spots with a capillary and let them dry. Repeated small applications with drying can concentrate a dilute sample while avoiding a large spreading spot.

Stand the plate in a covered developing chamber with solvent below the baseline. If the starting spots are immersed, sample can dissolve directly into the reservoir instead of travelling up the plate. Avoid touching or scraping the stationary coating.

The cover helps establish an atmosphere saturated with solvent vapour, reducing evaporation from the developing plate. It also limits solvent loss. Do not describe a TLC plate as paper that needs a lid simply to hold it upright.

Develop the plate and reveal the spots

Allow the solvent to rise by capillary action and remove the plate before the front reaches the top. Mark the solvent front immediately in pencil before it evaporates. Dry the plate as directed, then use an appropriate visualisation method.

Some samples are naturally coloured. Suitable compounds can be viewed using a UV lamp and the correct plate; other colourless samples need a locating reagent. Ninhydrin is useful for amino acids. A UV lamp is not a substance that can be sprayed onto a plate. Use eye protection and the approved viewing enclosure for UV work.

Mark the centre of each spot. Compare the mixture with standards developed on the same plate where possible. Matching solvent, stationary phase and temperature matters because Rf is not an immutable property of the molecule.

Calculate Rf from two distances

Rf = distance travelled by the centre of the substance spot from the baseline ÷ distance travelled by the solvent front from the same baseline. The units cancel. For an ordinary valid developed plate, Rf lies between 0 and 1.

Original example: the solvent travels 8.0 cm and a spot centre travels 5.2 cm. Rf = 5.2/8.0 = 0.65. Measuring to the upper edge of a broad spot, or measuring from the bottom of the plate, changes the result incorrectly.

A spot with Rf 0.30 has travelled 2.4 cm when the front has travelled 8.0 cm. If the plate develops to a different distance under otherwise equivalent conditions, use the ratio; do not expect the raw distance alone to identify a substance.

Interpret standards, mixtures and purity carefully

Original teaching data: standards P and Q have Rf values 0.25 and 0.65. An unknown developed alongside them has spots at 0.25, 0.40 and 0.65. The result supports the presence of components behaving like P and Q, plus at least one other detected component. It does not prove the identities uniquely.

Different substances can co-elute or overlap, producing one observed spot. Some components may not be visible with the chosen detection method. One spot is therefore consistent with purity under those conditions, not absolute proof of a pure substance. More visible spots indicate multiple detected components, assuming the sample has not changed during the analysis.

If all spots stay at the origin or travel with the front, adjust the mobile-phase composition appropriately and repeat. The aim is separated, measurable spots, not simply the largest possible Rf. Overloading, a submerged baseline, an unmarked front or a tilted solvent front can make interpretation unreliable.

As an optional extension, two-dimensional TLC uses one solvent, dries the plate, rotates it by 90° and develops it with a different solvent. Components that overlapped in the first direction may separate in the second. This does not guarantee that every component will be resolved.

Connect the practical step to its reason

AQA June 2023 Paper 3 Q03.4–03.7 links the cover, separation mechanism, locating agent and Rf calculation to an amino-acid mixture. The report explains why a complete two-phase account and a suitable developing agent matter. These are contextual lessons, not a claim that every TLC question has the same marking points.

An explanation such as “put on a lid for accuracy” is incomplete. State that the cover retains solvent vapour, helps saturate the chamber atmosphere and reduces evaporation from the plate. Similarly, say what a drying or visualisation step accomplishes.

Quick checks

Original Finesse questions. Reveal the indicative worked solutions after attempting each question; these are not official AQA mark allocations.

Sources