Chromatography Practice Questions

Chromatography questions appear in almost every chemistry course, from simple “which dyes are in this ink?” problems to reading gas chromatograms. These eighteen questions start with the basics and work up. Try each one before reading the answer. For the theory, see chromatography explained, paper chromatography and thin-layer chromatography.

Part A: The basics

1. In chromatography, what are the two phases, and what does each do?

Answer: The stationary phase stays still (the paper, the silica on a TLC plate, or the packing in a column). The mobile phase moves through or over it (the solvent, or a gas in GC). Substances separate because each one divides its time differently between the two phases.

2. Why is the starting line drawn in pencil rather than ink?

Answer: Pencil (graphite) doesn’t dissolve in the solvent, so it won’t move up the paper. An ink line would dissolve and separate, adding extra spots that confuse the result.

3. Why must the starting line be above the level of the solvent in the tank?

Answer: If the spots were below the solvent surface, they would dissolve into the solvent in the tank instead of being carried up the paper.

4. Why is a lid placed on the chromatography tank?

Answer: It keeps the air inside saturated with solvent vapour. This stops solvent evaporating from the paper as it rises, giving an even solvent front and more reproducible Rf values.

Part B: Rf calculations

Rf = distance moved by the substance ÷ distance moved by the solvent front, both measured from the starting line. See Rf value calculations.

5. The solvent front moves 9.0 cm. A spot moves 6.3 cm. Calculate Rf.

Answer: Rf = 6.3 ÷ 9.0 = 0.70

6. A dye has an Rf of 0.35 in a particular solvent. The solvent front moved 12.0 cm. How far did the dye travel?

Answer: Distance = 0.35 × 12.0 = 4.2 cm

7. A student calculates an Rf value of 1.25. What went wrong?

Answer: Rf can never be greater than 1, because a substance can’t travel further than the solvent carrying it. The student probably divided the solvent distance by the spot distance (the wrong way round), or measured from different points.

8. Why should the solvent front be marked as soon as the paper is removed from the tank?

Answer: The solvent evaporates quickly, and once it has dried, the position of the front can’t be seen. Without it, Rf can’t be calculated.

Part C: Interpreting chromatograms

9. A brown food colouring gives three spots with Rf values 0.20, 0.52 and 0.81. Reference dyes give: Red A 0.52, Yellow B 0.81, Blue C 0.35, Green D 0.20. Which dyes are in the brown colouring?

Answer: Red A (0.52), Yellow B (0.81) and Green D (0.20). Blue C (0.35) is not present.

10. One spot on a chromatogram stays on the starting line. Give two possible reasons.

Answer: The substance is insoluble in the solvent used, or it’s very strongly attracted to the stationary phase. Trying a different solvent may make it move.

11. A pure substance should give how many spots? What if a sample gives one spot in one solvent?

Answer: A pure substance gives one spot. But a single spot in one solvent doesn’t prove purity, because two substances might have the same Rf in that solvent. Running the sample in a second, different solvent is a better test.

12. Two inks are compared. Ink P gives spots at Rf 0.25 and 0.60; ink Q gives spots at 0.25, 0.60 and 0.85. A note found at a crime scene gives spots at 0.25 and 0.60. Which ink was used?

Answer: Ink P. Ink Q has an extra dye (Rf 0.85) that isn’t in the note. (In real forensic work, more tests would be done. See forensic chemistry.)

Part D: Polarity and TLC

13. On a silica TLC plate (a polar stationary phase) with a fairly nonpolar solvent, which moves further: a polar compound or a nonpolar compound? Explain.

Answer: The nonpolar compound moves further (higher Rf). The polar compound is attracted more strongly to the polar silica, so it spends more time stuck to the stationary phase. See polar vs nonpolar molecules.

14. All the spots on a TLC plate have run close to the solvent front. How should the solvent be changed?

Answer: Make the solvent less polar (for example, a higher proportion of hexane in a hexane–ethyl ethanoate mixture). The compounds will then interact relatively more with the silica and move less far, spreading the spots out.

15. Many organic compounds are colourless. Give two ways of seeing their spots on a TLC plate.

Answer: (1) Use a plate containing a fluorescent indicator and look under UV light; compounds that absorb UV show as dark spots. (2) Use a locating agent, such as iodine vapour, or a stain that reacts to give coloured spots (for example, ninhydrin for amino acids).

Part E: Gas chromatography and HPLC

16. In gas chromatography, what is the retention time, and how is it used?

Answer: The retention time is the time between injecting the sample and the peak for a component reaching the detector. Under the same conditions, a given compound always has the same retention time, so it’s compared with standards to identify components. See gas chromatography.

17. A gas chromatogram of a fuel shows three peaks with areas 20%, 50% and 30% of the total. What does this suggest about the mixture?

Answer: The peak area is proportional to the amount of each component (for similar compounds and a calibrated detector). So the mixture contains roughly 20%, 50% and 30% of the three components. For accurate percentages, each component’s detector response is calibrated with standards.

18. Why is HPLC, rather than GC, used to analyse the sugars in fruit juice?

Answer: Sugars are not volatile, and they decompose (caramelise) when heated strongly, so they can’t be vaporised for GC without first being chemically converted. HPLC works with liquid samples at or near room temperature. See HPLC.

Common mistakes to avoid

• Measuring distances from the bottom of the paper instead of the starting line.
• Measuring to the edge of a spot rather than its centre.
• Forgetting to mark the solvent front.
• Assuming one spot means pure.
• Confusing which compound moves further on a polar stationary phase.
• Writing Rf with units: it’s a ratio, so it has none.

Exam technique

• In “describe how” questions, list equipment and steps in order: pencil baseline, small concentrated spots, solvent below baseline, lid, remove before the solvent reaches the top, mark the front, dry, locate spots, measure and calculate Rf.
• In “explain” questions, refer to the two phases and to attraction or solubility differences.
• In data questions, show the division clearly and give Rf to two decimal places.

Key takeaways

• Separation depends on how each substance distributes between the stationary and mobile phases.
• Rf = spot distance ÷ solvent distance, always ≤ 1, no units.
• Matching Rf values with references (in more than one solvent) identifies components.
• On polar silica, nonpolar compounds travel further.
• GC identifies by retention time and quantifies by peak area; HPLC suits non-volatile or heat-sensitive compounds.