Chromatography: two phases pull a mixture apart
Definition
Chromatogram
The pattern of separated spots produced by a chromatography experiment.
Definition
Mobile phase
The phase that moves through the stationary phase during chromatography, carrying dissolved substances with it; in paper chromatography it is the solvent.
Definition
Stationary phase
The phase that does not move during chromatography; in paper chromatography it is the chromatography paper.
- Paper chromatography separates the substances in a mixture using two phases that compete for each substance.
- The stationary phase is the chromatography paper, which stays still.
- The mobile phase is the solvent that soaks up the paper and carries dissolved substances with it.
- Each substance keeps swapping between being dissolved in the moving solvent and being held on the paper.
- A substance that is more soluble in the solvent and less strongly attracted to the paper spends more time moving, so it travels further.
- The finished pattern of separated spots is a chromatogram.
Key Idea
- Separation happens because each substance strikes a different balance between the moving solvent and the still paper.
- Distance travelled is a fingerprint: in the same conditions a substance always moves the same way.
Reading a chromatogram: one spot means pure, more spots mean a mixture
- A pure substance produces a single spot that stays as one spot whichever solvent is used.
- A mixture produces two or more spots because its substances travel different distances.
- Counting the spots tells you the smallest number of substances present in the sample.
- If a spot in a mixture lines up with a known substance at the same height, they may be the same substance.
Common Mistake
- Always draw the start line in pencil: ink is a mixture that would dissolve and run up the paper, ruining the result.
- One spot in a single solvent is not full proof of purity; a pure result should stay a single spot in a different solvent too.
Rf values: turning distances into a number that identifies a substance
Definition
Rf value
The distance moved by a substance divided by the distance moved by the solvent, both measured from the origin line; it has no units and is always between 0 and 1.
Definition
Origin line
The pencil line on which the samples are placed at the start of paper chromatography.
Definition
Solvent front
The furthest point reached by the solvent as it moves through the stationary phase.
- An Rf value compares how far a substance travels with how far the solvent travels.
- The formula is Rf=distance moved by substancedistance moved by solventR_f = \dfrac{\text{distance moved by substance}}{\text{distance moved by solvent}}Rf=distance moved by solventdistance moved by substance, with both distances measured from the origin line.
- Measure the substance distance to the centre of its spot, and the solvent distance to the solvent front.
- An Rf value has no units and always lies between 000 and 111.
- In the same solvent and paper a substance always gives the same Rf value, so a known value can be used to identify it.
- Worked example: a spot moves 24 mm24\,\text{mm}24mm while the solvent moves 60 mm60\,\text{mm}60mm, so Rf=2460=0.40R_f = \frac{24}{60} = 0.40Rf=6024=0.40.
Practical
Separating coloured substances by paper chromatography
- Draw a straight origin line in pencil across the chromatography paper, about 2 cm2\,\text{cm}2cm up from the bottom short edge.
- Mark five spots along the line, each at least 0.5 cm0.5\,\text{cm}0.5cm from the edges.
- Using a fresh capillary tube for each, add a small spot of the four known colourings AAA, BBB, CCC, DDD and the unknown mixture UUU, keeping every spot 2 to 3 mm across, and let them dry.
- Pour the solvent (water) into a beaker to a depth of no more than 1 cm1\,\text{cm}1cm.
- Hang the paper from a spill so its bottom edge dips in but the origin line stays above the solvent, and the sides do not touch the beaker walls.
- Leave the beaker undisturbed until the solvent has risen about three quarters of the way up the paper.
- Remove the paper, immediately mark the solvent front in pencil, and hang it up to dry.
- Measure the distance from the origin line to the solvent front, and to the centre of each spot, then work out each RfR_fRf value.
- Match the unknown UUU to the known colourings by comparing spot heights and RfR_fRf values.
Getting trustworthy results: technique that protects your chromatogram
- Keep the origin line and the spots above the solvent; if they start underwater the colours wash straight into the beaker.
- Let each spot dry and keep it small so the separated spots stay tight and do not overlap.
- Do not move the beaker while the solvent rises, or the front becomes uneven and the Rf values are wrong.
- Mark the solvent front the moment you lift the paper, because it dries and becomes impossible to see.
- Always measure to the centre of a spot so repeats can be compared fairly.
Self review
- What are the stationary phase and the mobile phase in paper chromatography?
- Why must the start line be drawn in pencil?
- How can a chromatogram show whether a substance is pure or a mixture?
- Write the equation for an Rf value and state its possible range.
- A spot moves 30 mm and the solvent moves 75 mm. What is the Rf value?
