Experiment 2: Leaf Chromatography — Pigments in Leaves

A leaf looks green, but is green the only pigment inside it? Chromatography answers that by separating a mixture: a solvent climbs a strip of paper by capillary action and carries each pigment a different distance, depending on how strongly that pigment sticks (adsorbs) to the paper and how well it dissolves in the solvent. The Virtual Lab: Paper Chromatography — Pigment Separation Lab on this page runs the separation for you; here you will run the bench version with spinach, interpret the bands, and quantify them with the Rf value.

The science behind the practical

Leaves contain several pigments, each with a job. Chlorophyll a (blue-green) and chlorophyll b (yellow-green) absorb light for photosynthesis. Carotenes (orange) and xanthophylls (yellow) are accessory pigments that widen the range of light captured and protect the leaf from damage. In autumn, chlorophyll breaks down first, so the yellows and oranges that were there all along become visible.

Separation works because the pigments differ in polarity. Non-polar carotene barely sticks to the paper and dissolves well in the solvent, so it travels furthest. The chlorophylls stick more strongly and lag behind. The Rf (retention factor) for a band is: Rf = distance moved by the pigment ÷ distance moved by the solvent front. Rf is always between 0 and 1 and is a fingerprint: the same pigment in the same solvent and paper gives the same Rf.

Variables, controls and fair testing

Running the investigation well

Mash the leaves in rubbing alcohol to extract the pigments — grinding thoroughly matters, because a pale extract gives faint bands. Draw the origin line in pencil (ink would chromatograph too), spot the extract on it several times, letting it dry between spots to concentrate it. Stand the strip so the solvent level is *below* the spot; if the spot dips into the solvent, your pigments dissolve into the cup instead of climbing. In the simulator, watch which band separates first and note the order of the bands — the order, not the colour brightness, is what identifies them. The moment you remove the strip, mark the solvent front in pencil before it evaporates.

Safety: rubbing alcohol is flammable — no flames nearby, and work in a ventilated space.

Worked example: a sample investigation

A spinach extract ran until the solvent front reached 8.0 cm from the origin. Four bands appeared:

Analysing the sample data

Step 1 — calculate each Rf. Divide each distance by 8.0 cm:

• Band 1: 7.6 ÷ 8.0 = 0.95 → carotene (travels furthest, sticks least)
• Band 2: 5.6 ÷ 8.0 = 0.70 → xanthophyll
• Band 3: 4.0 ÷ 8.0 = 0.50 → chlorophyll a
• Band 4: 2.8 ÷ 8.0 = 0.35 → chlorophyll b (sticks most)

Step 2 — interpret. The green colour of the whole leaf hides the orange and yellow pigments; chromatography proves at least four pigments were present in a leaf that looked one colour. For a purple leaf run in the same setup, a student found only two bands, one with Rf = 3.2 ÷ 8.0 = 0.40, matching neither chlorophyll exactly — evidence for a different pigment (an anthocyanin) dominating.

Step 3 — describe the graph. A bar chart of Rf by band falls in a staircase from 0.95 down to 0.35. The even spacing shows the four pigments differ steadily in how strongly they adsorb to paper — the physical reason the separation works.

Conclusion and evaluation

Conclusion: a green leaf contains at least four pigments — carotene, xanthophyll, chlorophyll a and chlorophyll b — identified by their characteristic Rf values (0.95, 0.70, 0.50, 0.35 in this solvent).

Evaluation: Rf values are only comparable within the same solvent, paper and temperature; quoting them without those conditions is meaningless. Faint bands risk being missed, so the count "four" is a minimum. Improvements: concentrate the spot more, run the strip longer for wider separation, and repeat with a second leaf species for comparison.

Safety and ethics

Rubbing alcohol is flammable and an irritant: no naked flames, ventilate the room, and never taste extracts. Plant material only, so no animal-ethics issues arise.

Common mistakes

• Using pen for the origin line — the ink separates and contaminates the results.
• Letting the solvent cover the spot, washing the pigments into the cup.
• Forgetting to mark the solvent front immediately, making Rf impossible to calculate.
• Identifying bands by colour alone without Rf values — colours vary with concentration; Rf is the evidence.

Exam Experience

Question 1 — Analysis (4 marks). In a new run, the solvent front reaches 10.0 cm. Bands are found at 9.2, 6.8, 4.9 and 3.1 cm. Calculate the Rf of each band and identify the band most likely to be carotene, giving your reason. *Scoring guide:* Rf values 0.92, 0.68, 0.49, 0.31 (1 mark for all four, method shown). Carotene = band at 9.2 cm / Rf 0.92 (1 mark) because it travelled furthest, meaning it adsorbs least to the paper and is most soluble in the solvent (1 mark). Final mark for consistent use of the same solvent front value throughout.

Question 2 — Evaluation and design (4 marks). A student's strip shows only one green smear instead of separate bands. Give two likely causes and, for each, the change that fixes it. Then state one variable that must be controlled when comparing two leaves. *Scoring guide:* Causes/fixes (1 mark each pair, max 2 pairs): extract too dilute → spot repeatedly and dry between spots; solvent covered the spot → lower the solvent level; run too short → allow more time; strip touched the wall → centre the strip. Controlled variable (1 mark): same solvent/paper/running time/starting height.

Multiple choice 1. A pigment moves 3.0 cm while the solvent moves 7.5 cm. Its Rf is: A) 0.04 B) 0.40 C) 2.5 D) 4.5. *Key: B.* Rf = 3.0 ÷ 7.5 = 0.40. C divides the wrong way; D subtracts; A misplaces the decimal.

Multiple choice 2. The pigment that travels furthest on the paper is the one that: A) is heaviest B) sticks most to the paper C) sticks least to the paper and dissolves best in the solvent D) is present in the greatest amount. *Key: C.* Travel distance reflects adsorption and solubility, not mass or quantity (A, B, D wrong; B describes the band that stays nearest the origin).

Going deeper: what Rf can and cannot tell you

An Rf value is a fingerprint only inside a defined system: same paper, same solvent, same temperature. Change the solvent and the whole pattern can shift — a more polar solvent drags the chlorophylls further, for instance — which is why published Rf tables always name their conditions. In the worked example, a longer run with the solvent front at 9.0 cm gave a yellow band at 6.3 cm: Rf = 6.3 ÷ 9.0 = 0.70, matching xanthophyll from the first run exactly. Two different runs, two different distances, one identical Rf — that reproducibility is precisely what makes Rf an identification tool rather than a rough description.

Chromatography also scales up beyond leaves. The same principle — a mixture carried past a material that holds its components back by different amounts — separates the dyes in ink (forensic scientists use it to compare pen inks on questioned documents), the colourings in food, and, in industrial columns, the components of medicines. Thin-layer chromatography on silica plates is the direct bench descendant of your coffee-filter strip.

A subtle point worth carrying into the exam: the bands' *order* is a property of the pigments' chemistry, but their *brightness* is a property of amount. A faint carotene band does not mean carotene "ran badly"; it means there was less of it in the extract. Never read quantity conclusions into a separation pattern without a standard of known concentration beside it.

A second look: how trustworthy is an Rf?

Three groups in one class ran the same spinach extract in the same solvent and measured chlorophyll a at Rf 0.48, 0.50 and 0.52. The mean is (0.48 + 0.50 + 0.52) ÷ 3 = 0.50, with a range of 0.04 — tight agreement, because Rf divides out the biggest accidental variable (how far each strip happened to run). That is the deep reason ratios rule this course: Rf here, percentage change in osmosis, percentage cover in the mould race. Absolute distances would have scattered far more between groups whose solvent fronts stopped at different heights.

The same data teach the opposite lesson about faint bands. Two groups reported four bands; the third, using a paler extract, reported three — carotene, the faintest in their run, sat below what the eye could pick out. Their result was not *wrong*; it was less sensitive. In practical science, "we found four pigments" always silently means "four pigments above our detection limit", and stating the limit is part of stating the result.

Final check: the autumn leaf, caught in the act

Run an autumn (yellowing) leaf in the same system — solvent front again 8.0 cm — and the chromatogram tells the seasonal story in numbers. Bands appear at 7.5 cm (orange, Rf = 7.5 ÷ 8.0 = 0.94), 5.4 cm (yellow, Rf = 0.68), then only a faint blue-green smudge at 1.6 cm (Rf = 0.20) and a trace at 0.8 cm (Rf = 0.10). Matching within measurement error, the top two bands are the familiar carotene (0.95) and xanthophyll (0.70) — barely diminished. The chlorophylls, by contrast, have collapsed in amount, and their smudged, shifted remnants are breakdown products, not the originals. The leaf did not manufacture orange and yellow for autumn; it *stopped manufacturing green*, and the colours that were masked all summer simply became the last ones standing. A separation you ran in spring predicts the chemistry of a season you have not reached yet — that is what a good measurement is for.