Every chromatography method shares a stationary phase and a mobile phase. Thin-layer chromatography gives Rf values that identify spots; gas-liquid chromatography gives retention times that identify peaks and peak areas that tell you how much of each component is present.
All chromatography separates a mixture between a stationary phase, which stays put, and a mobile phase, which moves through it. A component that interacts more strongly with the stationary phase moves more slowly. In thin-layer chromatography (TLC) the stationary phase is silica or alumina on a plate and separation is mainly by adsorption; each spot has an Rf value = distance moved by the spot ÷ distance moved by the solvent front. In gas-liquid chromatography (GLC) an inert carrier gas carries the vapourised sample through a column coated with a non-volatile liquid, and separation is by partition. Each component has a characteristic retention time, and the area under each peak is proportional to the amount of that component, so relative peak areas give the percentage composition.
Fig. 1: measuring Rf values on a developed TLC plate
Section 01
Two phases, one separation.
Each method below has the same two ingredients. What changes is what the stationary phase is made of and how strongly each component sticks to it.
Method
Stationary phase
Mobile phase
Main separation process
paper
water held in the cellulose fibres
a liquid solvent
partition between two liquids
thin-layer (TLC)
thin layer of silica (SiO2) or alumina on a glass, metal or plastic sheet
a liquid solvent
adsorption onto the solid surface
gas-liquid (GLC)
non-volatile liquid coated on a solid support inside a long thin column
an inert carrier gas, e.g. helium or nitrogen
partition between gas and liquid
Partition: a component distributes itself between two phases according to its relative solubility in each.
Adsorption: a component is held on the surface of a solid by intermolecular forces; polar compounds stick more strongly to polar silica, so they move more slowly.
Locating colourless spots: shine UV light on a plate containing a fluorescent indicator (spots show up dark), expose the plate to iodine vapour (brown spots), or spray ninhydrin and warm, which turns amino acids purple.
Good practice: draw the baseline in pencil (ink would itself separate), keep the solvent level below the baseline, cover the tank so the air stays saturated with solvent vapour, and mark the solvent front as soon as the plate is removed.
Stage 1 · Learn
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On a TLC plate a spot moves 3.0 cm and the solvent front moves 7.5 cm. What is the Rf value?
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What is the mobile phase in gas-liquid chromatography?
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Why is the baseline on a chromatography plate drawn in pencil?
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How are amino acid spots located on a chromatogram?
Section 02
Rf values on the plate.
An Rf value is a ratio, so it has no units and always lies between 0 and 1. It is only characteristic of a compound for a given solvent and stationary phase.
Spot
Distance moved by spot / cm
Distance moved by solvent front / cm
Rf
A
2.0
8.0
0.250
B
5.6
8.0
0.700
C
3.9
6.5
0.600
Measure from the baseline to the centre of the spot, and from the baseline to the solvent front.
Identifying a component: run the unknown beside pure standards on the same plate. A spot with the same Rf as a standard, in the same solvent, is probably that compound; running a second solvent gives stronger evidence.
Working backwards: a compound with Rf 0.450 in a run where the front moves 9.0 cm travels 0.450 × 9.0 = 4.05 cm.
Low Rf means strong attraction to the stationary phase (for silica, a more polar compound); high Rf means the compound spends more time in the mobile phase.
Section 03
Gas-liquid chromatography and peak areas.
In GLC the detector draws a chromatogram: one peak per component, plotted against time from injection.
Peak
Retention time / min
Peak area / arbitrary units
Percentage
ethanol
2.1
18
18 ÷ 60 × 100 = 30.0%
propan-1-ol
3.4
30
30 ÷ 60 × 100 = 50.0%
butan-1-ol
5.2
12
12 ÷ 60 × 100 = 20.0%
Retention time is the time from injection to the peak maximum. It depends on the column, its temperature and the carrier gas flow rate, so standards must be run under identical conditions.
Why the order? A more volatile component, or one less soluble in the liquid stationary phase, spends more time in the gas and comes out first.
Percentage composition = peak area ÷ total of all peak areas × 100, assuming the detector responds equally to each component.
Limitation: similar compounds can have almost the same retention time, and a new compound has no standard, so GLC is often linked to a mass spectrometer (GC-MS) to identify each peak.
Examiner trap
Rf is spot distance ÷ solvent front distance, never ÷ the length of the plate, and both are measured from the baseline. In GLC the amount of a component is given by the peak area, not its height, and the percentage uses the total of all peak areas. A long retention time means strong interaction with the stationary phase, not a large amount of substance.
Stage 2 · Exam
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A GLC chromatogram of a mixture of three esters has peak areas of 42, 87 and 21 units. What is the percentage of the component with area 87?
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In TLC on silica, compound P has Rf 0.15 and compound Q has Rf 0.80 in the same solvent. Which statement is correct?
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A compound has Rf 0.450 in a particular solvent. The solvent front moves 9.0 cm. How far from the baseline is the spot?
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Two components of a mixture give peaks with the same retention time on one GLC column. What is the best next step to identify them?
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