Colourless substances
Definition
Locating agent
A substance applied to a dried chromatogram that makes the spots of colourless substances visible.
- Many substances, such as amino acids and sugars, are colourless, so their spots cannot be seen on the paper after the solvent has run.
- They are separated in exactly the same way as coloured substances, using a pencil baseline and a suitable solvent whose level is below the baseline.
- After the run, the paper is dried and treated with a locating agent, usually by spraying it evenly over the paper.
- The locating agent reacts with the separated substances to form coloured products, so each substance appears as a visible spot.
- The solvent front is marked in pencil as soon as the paper is removed, before the solvent evaporates and the wet edge disappears.
- Each spot is outlined in pencil as soon as it appears, because some of the coloured spots fade over time.
Practical
Separating a mixture of amino acids
- Aim: to separate a colourless mixture of amino acids and identify them using a locating agent.
- Apparatus: chromatography paper, a pencil, a ruler, capillary tubes, a chromatography tank or tall beaker with a lid, a suitable solvent, a spray bottle of locating agent, a fume cupboard, protective gloves, an oven or hairdryer, the unknown mixture and solutions of known amino acids.
- Method, running the chromatogram:
- Wear gloves or hold the paper only by its edges, because amino acids from your fingertips would show up as extra spots.
- Draw a pencil baseline about 2 cm2\ \text{cm}2 cm from the bottom and put small spots of the mixture and of each known amino acid along it.
- Stand the paper in the tank with the solvent level below the baseline, then put the lid on.
- Remove the paper when the solvent is close to the top and mark the solvent front in pencil straight away.
- Method, locating the spots:
- Dry the paper completely in a fume cupboard.
- Still in the fume cupboard, spray the locating agent lightly and evenly over the whole paper.
- Warm the paper gently in an oven or with a hairdryer until coloured spots appear.
- Draw round each spot in pencil and mark its centre.
- Results: measure the distance from the baseline to the centre of each spot, and from the baseline to the solvent front.
- Conclusion: each spot from the mixture that is level with the spot of a known amino acid is that amino acid.
- Watch out: if the solvent front is not marked immediately, the solvent evaporates and the distance it travelled cannot be measured.
- Safety: many locating agents and chromatography solvents are toxic or flammable, so spray only in a fume cupboard, wear gloves and eye protection, and keep solvents away from flames.
RfR_fRf values
Definition
Rf value
The distance moved by a substance divided by the distance moved by the solvent in the same chromatogram.
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An RfR_fRf value compares how far a substance has travelled with how far the solvent has travelled in the same time.
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It is calculated with the equation:
Rf=distance travelled by substancedistance travelled by solvent R_f = \frac{\text{distance travelled by substance}}{\text{distance travelled by solvent}} Rf=distance travelled by solventdistance travelled by substance -
The distance travelled by the substance is measured from the baseline to the centre of its spot.
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The distance travelled by the solvent is measured from the baseline to the solvent front.
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Both distances are in the same unit, so the units cancel and an RfR_fRf value has no unit.
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A substance can never travel further than the solvent that carries it, so every RfR_fRf value lies between 000 and 111.
- A substance that stays on the baseline has an RfR_fRf value of 000.
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A given substance always has the same RfR_fRf value when it is run in the same solvent on the same type of paper.
- This means you can identify an unknown substance by comparing its RfR_fRf value with a table of known values, even when no known sample was run on the same paper.
Example
Identifying an amino acid from its RfR_fRf value
- On a chromatogram, the solvent front is 8.0 cm8.0\ \text{cm}8.0 cm above the baseline and the centre of an unknown spot is 5.2 cm5.2\ \text{cm}5.2 cm above the baseline.
- A data table for the same solvent gives RfR_fRf values of 0.300.300.30 for amino acid P, 0.650.650.65 for amino acid Q and 0.800.800.80 for amino acid R.
- The unknown is amino acid Q, because its RfR_fRf value matches.
- The answer is given to two significant figures, matching the two significant figures in the measured distances.
Exam technique
Calculating RfR_fRf values
- Measure from the baseline, never from the bottom edge of the paper.
- Measure to the centre of a spot, not to its top or bottom edge.
- Check that your answer is less than 1, because a value greater than 1 means the distances have been put the wrong way round.
- Give the RfR_fRf value as a decimal with no unit.
Common Mistake
- Do not measure the solvent distance to the top of the paper, because the solvent front is usually lower.
- Do not say that the locating agent separates the substances, because the solvent separates them and the locating agent only makes them visible.
- Do not compare an RfR_fRf value with data obtained using a different solvent.
Self review
- Why is a locating agent needed for some chromatograms?
- Why must the solvent front be marked as soon as the paper is removed?
- State the equation for RfR_fRf.
- Why can an RfR_fRf value never be greater than 1?
- A spot travels 3.0 cm3.0\ \text{cm}3.0 cm while the solvent travels 7.5 cm7.5\ \text{cm}7.5 cm, so what is its RfR_fRf value?
