How chromatography separates a mixture
Chromatography has two phases: a stationary phase固定相 that stays put (paper, a silica plate, the packing in a column) and a mobile phase流动相 that flows past it (the solvent, or eluent). Each component keeps moving back and forth between the two:
- the more strongly it interacts with the stationary phase, the longer it stays and the more slowly it moves;
- the more strongly it interacts with the mobile phase, the faster it’s carried along.
Components with different intermolecular forces move at different speeds and separate. Paper (cellulose) and silica are both covered in –OH groups, so they are polar stationary phases.
Rf values
- Rf is between 0 and 1; both distances are measured from the starting line.
- With the same stationary phase, solvent and temperature, a substance always has the same Rf, so comparing with standards identifies it.
- Column chromatography has no Rf; what matters is the order of elution: the component that interacts more weakly with the stationary phase comes out first.
Practical points
- Draw the starting line in pencil (ink would dissolve in the solvent and separate), and keep it above the solvent level, or the sample dissolves straight into the solvent.
- Keep the spots small and concentrated; remove the paper when the front nears the top and mark the solvent front right away.
Example 1: calculating Rf and identifying a substance
On a silica TLC plate, the solvent front is 7.5 cm from the starting line and the unknown’s spot has moved 4.8 cm. On the same plate, standards A and B have Rf values of 0.64 and 0.30. Which could the unknown be?
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Rf of the unknown.
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Compare with the standards. On the same plate with the same solvent, the unknown has the same Rf as A.
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Conclusion. The unknown could be A. To be more confident, run it again with a different solvent and check whether the two Rf values still match.
Example 2: explaining the order with intermolecular forces
Naphthalene (C₁₀H₈) and 2-naphthol (C₁₀H₇OH) are separated on a silica TLC plate with hexane as the solvent. Which has the larger Rf? Explain.
- Compare the two substances. Naphthalene contains only C and H; it is nonpolar, with only dispersion forces. 2-Naphthol has an extra –OH group and can hydrogen bond.
- Interaction with the stationary phase. Silica’s surface has Si–OH groups. 2-Naphthol hydrogen bonds to them and is held more tightly; naphthalene has only weak dispersion forces with silica.
- Interaction with the mobile phase. Nonpolar hexane carries nonpolar naphthalene along more easily.
- Conclusion. Naphthalene has the larger Rf; 2-naphthol stays closer to the starting line.
- Silica plate with hexane: switch to a more polar compound
It interacts more strongly with polar silica and moves less.
- Silica plate: ethyl acetate is added to make the solvent more polar; consider a polar compound
The mobile phase can now pull the polar compound along more effectively.
- Nothing else changes, but the plate develops longer so the solvent front goes farther
The spot and front move proportionally farther, so the ratio stays the same.
- Use a longer strip of the same paper
Rf is a ratio and doesn’t depend on the strip’s length.
- Reversed-phase chromatography (nonpolar stationary phase, polar mobile phase): the component becomes more polar
It interacts less with the nonpolar stationary phase and more with the polar mobile phase.
- Reversed phase: the fraction of organic solvent in the mobile phase increases (less polar); consider a nonpolar component
The mobile phase dissolves the nonpolar component better and carries it along.
- Paper with 70% isopropanol: switch to a more polar dye
The solvent is less polar, so a more polar dye stays on the polar paper more.
- Paper with salt water: switch to a more polar dye
The polar solvent dissolves a polar dye better and carries it farther.
Common mistakes
Mistake 1: “A large Rf means small, light molecules.” Paper and thin-layer chromatography separate by the relative strength of interactions with the two phases, not by molecular size.
Mistake 2: “A substance has the same Rf under any conditions.” Rf depends on the stationary phase, the solvent and the temperature. Comparing Rf values only makes sense under the same conditions.
Mistake 3: “Polar substances always move slowly.” It depends on both phases. With a polar stationary phase and a less polar solvent, polar substances move slowly; with a very polar solvent, or in reversed-phase chromatography, the order flips.
Quick check
A spot moves 3.0 cm and the solvent front moves 6.0 cm. What is Rf?
Rf = 3.0 ÷ 6.0 = 0.50.
What is the possible range of Rf?
A spot can’t move past the solvent front.
Why is the starting line drawn in pencil?
Graphite doesn’t dissolve in the solvent, so it doesn’t move.
Why must the starting line be above the solvent level?
A sample under the solvent washes away instead of moving up the paper.
An unknown and a standard have the same Rf under the same conditions. The most reasonable conclusion is:
Different substances can have the same Rf by coincidence.
The basic reason chromatography separates a mixture is that:
Different interactions mean different distributions between the phases, and so different speeds.
AP-style practice
A student spots a spinach-leaf extract on a silica TLC plate and develops it with a hexane–acetone mixture (mostly hexane). The solvent front is 7.2 cm from the starting line, with these results:
| Pigment | Color | Distance (cm) |
|---|---|---|
| β-Carotene | Orange-yellow | 6.8 |
| Chlorophyll a | Blue-green | 3.1 |
| Chlorophyll b | Yellow-green | 2.3 |
(a) Calculate the Rf values of β-carotene and chlorophyll a.
(b) β-Carotene (C₄₀H₅₆) is a hydrocarbon; chlorophyll molecules contain C=O groups, ester groups, and N atoms bonded to Mg. Use intermolecular forces to explain why β-carotene’s Rf is much larger.
(c) If the proportion of acetone in the solvent is increased, how will the Rf of chlorophyll a change? Explain.
(d) If the pigments are separated in a column with the same silica and the same eluent, which comes out first?
(e) Chlorophyll b differs from chlorophyll a in that a –CH₃ group in chlorophyll a is replaced by –CHO in chlorophyll b. Explain why chlorophyll b has the smaller Rf.
Model answer and scoring
(a) β-Carotene: 6.8 ÷ 7.2 = 0.94; chlorophyll a: 3.1 ÷ 7.2 = 0.43.
(b) β-Carotene is nonpolar, so it has only weak dispersion forces with polar silica (whose surface has Si–OH groups), and the mostly hexane solvent carries it along easily. Chlorophyll has polar groups, giving dipole–dipole attractions (and hydrogen bonds to Si–OH) with silica, so it is held more tightly and moves less.
(c) It increases. Acetone is more polar than hexane; a more polar solvent interacts more strongly with chlorophyll’s polar groups and competes better with the silica for it, so chlorophyll moves farther.
(d) β-Carotene comes out first, because it interacts most weakly with the stationary phase.
(e) The C=O in –CHO is polar, making chlorophyll b more polar than chlorophyll a; its dipole–dipole attractions to silica are stronger, so it moves less.
Scoring: (a) both Rf values; (b) comparing the forces each has with the polar stationary phase (dispersion vs dipole–dipole or hydrogen bonding); (c) increases, explained by the solvent’s polarity; (d) β-carotene; (e) explained by the polarity of C=O.
Summary
- In chromatography, components divide between the stationary and mobile phases; the more strongly a component interacts with the stationary phase, the more slowly it moves.
- Rf = spot distance ÷ solvent front distance, both from the starting line; only Rf values under the same conditions can be compared.
- To explain an order, compare the intermolecular forces between each component and the two phases; changing the polarity of the solvent or stationary phase changes both the order and the Rf values.
AP tip: A chromatography explanation needs three things: the component’s structure (whether it has polar groups), the type of force it has with the stationary phase, and therefore whether it moves more or less.
Sources: the Rf values and distances in the simulation and practice are illustrative.