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TLC and Rf Values Worksheet

Lab activitiesGrades 9–12 · GCSE / A-level · IB · AP · undergraduate organic lab15 items · answer key included

A printable thin layer chromatography (TLC) worksheet with answers. Students read distances from drawn TLC plates — baseline, solvent front and spots, all labelled in millimetres — and calculate Rf values (distance moved by spot ÷ distance moved by solvent front) to 2 decimal places. They then identify the components of a mixture by comparing Rf values with standards, and answer questions on polarity, the stationary and mobile phases, and how to choose a better eluent. The same Rf calculation skills apply to paper chromatography for GCSE chemistry, as well as A-level, IB, AP and first-year organic chemistry labs.

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TLC and Rf Values Worksheet

Name Class Date

Part A — Calculate Rf values

Plate 1 shows a mixture M separated on a silica gel TLC plate. Calculate the Rf of each spot to 2 decimal places.

baseline (pencil)solvent frontM1: 20 mm2: 44 mm3: 68 mm80 mm
Plate 1 (silica; distances from baseline)
1.
SpotDistance moved by spot / mmDistance moved by solvent / mmRf
120800.25
244800.55
368800.85
2.
Which spot on Plate 1 is the most polar compound? Explain.
Spot 1 (Rf 0.25). It moved the shortest distance, so it is adsorbed most strongly by the polar silica stationary phase — it is the most polar.
3.
A spot moves 41 mm while the solvent front moves 64 mm. Calculate the Rf value.
Rf = 41 ÷ 64 = 0.64
4.
A compound has Rf = 0.35. The solvent front moved 60 mm. How far did the spot move?
Distance = 0.35 × 60 = 21 mm

Part B — Identify the components of a mixture

Plate 2: standards P, Q and R and an unknown mixture M were spotted on the same plate and developed together.

baseline (pencil)solvent frontPQRM301251125175 mm
Plate 2 (spot distances in mm)
5.
LaneSpot distance / mmRf
P300.40
Q120.16
R510.68
M (lower spot)120.16
M (upper spot)510.68
6.
Which standards are present in M? Which is absent?
M contains Q (Rf 0.16) and R (Rf 0.68); P (Rf 0.40) is absent.
7.
Why are the standards and the mixture run on the same plate rather than comparing with Rf values from a book?
Rf depends on the solvent, temperature, plate type and chamber saturation, so it is only reliable when samples are run side by side under identical conditions (co-spotting confirms identity).

Part C — Polarity and choice of solvent

Plates 3a and 3b: the same compound X was run on silica using hexane (3a) and 30 % ethyl acetate in hexane (3b).

baseline (pencil)solvent frontX7 mm70 mm
3a: hexane
baseline (pencil)solvent frontX35 mm70 mm
3b: 30 % EtOAc/hexane
8.
Calculate the Rf of X on plates 3a and 3b.
3a: 7 ÷ 70 = 0.10; 3b: 35 ÷ 70 = 0.50
9.
Explain why X moves further in eluent 3b.
Ethyl acetate is more polar than hexane. The more polar mobile phase competes with X for the polar silica surface, so X spends more time dissolved in the mobile phase and travels further (higher Rf).
10.
Which eluent is better for separating X from impurities? What Rf range should you aim for?
Eluent 3b — an Rf of about 0.2–0.6 gives good separation; at Rf 0.10 spots crowd near the baseline.
11.
Give two reasons why the baseline must be above the level of the solvent in the tank.
If the spots are below the solvent they dissolve into the solvent pool instead of moving up the plate, and the starting point would not be a fixed reference for measuring distances.
12.
The compounds are colourless. Give two ways to make the spots visible.
View under a UV lamp (254 nm) on a fluorescent-indicator plate — spots appear dark; or place the plate in an iodine chamber (brown spots) / dip in a stain such as KMnO4 or ninhydrin (for amino acids).

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Answer key: TLC and Rf Values Worksheet

Part A — Calculate Rf values

1.
SpotDistance moved by spot / mmDistance moved by solvent / mmRf
120800.25
244800.55
368800.85
2.
Spot 1 (Rf 0.25). It moved the shortest distance, so it is adsorbed most strongly by the polar silica stationary phase — it is the most polar.
3.
Rf = 41 ÷ 64 = 0.64
4.
Distance = 0.35 × 60 = 21 mm

Part B — Identify the components of a mixture

5.
LaneSpot distance / mmRf
P300.40
Q120.16
R510.68
M (lower spot)120.16
M (upper spot)510.68
6.
M contains Q (Rf 0.16) and R (Rf 0.68); P (Rf 0.40) is absent.
7.
Rf depends on the solvent, temperature, plate type and chamber saturation, so it is only reliable when samples are run side by side under identical conditions (co-spotting confirms identity).

Part C — Polarity and choice of solvent

8.
3a: 7 ÷ 70 = 0.10; 3b: 35 ÷ 70 = 0.50
9.
Ethyl acetate is more polar than hexane. The more polar mobile phase competes with X for the polar silica surface, so X spends more time dissolved in the mobile phase and travels further (higher Rf).
10.
Eluent 3b — an Rf of about 0.2–0.6 gives good separation; at Rf 0.10 spots crowd near the baseline.
11.
If the spots are below the solvent they dissolve into the solvent pool instead of moving up the plate, and the starting point would not be a fixed reference for measuring distances.
12.
View under a UV lamp (254 nm) on a fluorescent-indicator plate — spots appear dark; or place the plate in an iodine chamber (brown spots) / dip in a stain such as KMnO4 or ninhydrin (for amino acids).

How to use this worksheet

  1. Distances are printed on the plates so the sheet works at any print scale; for a measuring task, ask students to measure the plates with a ruler and compare to the labels.
  2. Use Part A as a starter on Rf calculations, then Part B as an identification problem like a real co-spot TLC.
  3. Part C links Rf to polarity on silica — a common stumbling block; discuss before a practical TLC of analgesics or plant pigments.
  4. Remind students that Rf has no units and must be between 0 and 1.
  5. Set the title, student lines and answer option on the left, then press Download PDF and choose “Save as PDF” (or print directly). Your choices are remembered on this device.

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Frequently asked questions

How do you calculate an Rf value?

Rf = distance travelled by the spot ÷ distance travelled by the solvent front, both measured from the baseline (to the centre of the spot). Rf has no units and lies between 0 and 1.

Does a polar compound have a high or low Rf on silica TLC?

Low. Silica gel is a polar stationary phase, so polar compounds adsorb more strongly and move less far; less polar compounds spend more time in the mobile phase and have higher Rf values.

What happens to Rf values if the eluent is made more polar?

All Rf values increase, because a more polar mobile phase competes better for the polar silica surface and carries compounds further up the plate.

Why is the TLC baseline drawn in pencil?

Graphite does not dissolve in the solvent or move up the plate, whereas ink contains dyes that would separate and confuse the chromatogram.

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