Chromatography of Plant Pigments

Separating chlorophyll and carotenoids using paper chromatography

Qualitative Analysis Intermediate (High School) 60 min ~$8.00

Objective

Separate and identify plant pigments using paper chromatography, and calculate Rf values for each pigment.

Background

Paper chromatography separates mixtures based on differential affinity for the stationary phase (paper) and mobile phase (solvent). Plant leaves contain multiple pigments: chlorophyll a (blue-green), chlorophyll b (yellow-green), xanthophylls (yellow), and carotenoids (orange). These pigments have different polarities, causing them to travel different distances up the paper as the solvent front advances.

Safety Warnings

  • Acetone and petroleum ether are highly flammable — no open flames
  • Work in a well-ventilated area or fume hood
  • Avoid inhaling solvent vapors
  • Petroleum ether is a skin irritant

Required PPE

goggles gloves lab_coat

Materials

  • Fresh spinach leaves (5–10 leaves)
    Or other dark green leaves
  • Acetone or isopropanol (50 mL)
    Extraction solvent
  • Petroleum ether (30 mL)
    Chromatography solvent
  • Chromatography paper (2 strips)
    Whatman No. 1 or equivalent
  • Sand (1 teaspoon)
    For grinding

Equipment

Mortar and pestle Beaker (250 mL) with watch glass cover Pencil (not pen) Ruler Capillary tube Scissors

Procedure

1

Grind spinach leaves with a pinch of sand and 5 mL acetone in a mortar until a deep green extract forms.

5 min Acetone is flammable
2

Draw a pencil line 2 cm from the bottom of the chromatography paper. Apply the extract using a capillary tube in a small spot. Let dry and reapply 3–4 times to concentrate.

10 min
3

Pour petroleum ether into the beaker to a depth of about 1 cm. Suspend the paper strip so the origin line is just above the solvent level.

5 min Work in fume hood
4

Cover the beaker with a watch glass and let the solvent rise by capillary action. Do not disturb until the solvent front is 1–2 cm from the top.

25 min
5

Remove the paper, immediately mark the solvent front with pencil. Let dry completely.

5 min
6

Identify the colored bands: carotenoids (orange, highest Rf), xanthophylls (yellow), chlorophyll a (blue-green), chlorophyll b (yellow-green, lowest Rf). Calculate Rf values for each band.

10 min

Expected Results

Four or more colored bands should be visible. Carotenoids (orange) travel farthest (Rf ≈ 0.9–0.95). Xanthophylls (yellow) at Rf ≈ 0.7. Chlorophyll a (blue-green) at Rf ≈ 0.5. Chlorophyll b (yellow-green) at Rf ≈ 0.4.

Cleanup

Allow all solvents to evaporate in the fume hood. Dispose of paper in general waste. Clean mortar and pestle thoroughly.

Details

Category
Qualitative Analysis
Difficulty
Intermediate (High School)
Duration
60 min
Est. Cost
$8.00
Steps
6
Materials
5

More Experiments

Anion Identification Tests 60 min Beilstein Test for Halogens 25 min Borax Bead Test for Metal Ions 45 min Cation Identification by Precipitation 90 min Flame Emission Spectroscopy 60 min Hard Water Testing with Soap Solution 40 min pH Indicator Rainbow 45 min Starch-Iodine Test for Carbohydrates 30 min Unknown Substance Identification Challenge 75 min

Frequently Asked Questions

What is the objective of Chromatography of Plant Pigments?
Separate and identify plant pigments using paper chromatography, and calculate Rf values for each pigment.
How difficult is Chromatography of Plant Pigments?
This experiment is rated as Intermediate (High School). It takes approximately 60 minutes to complete.
What safety precautions are needed for Chromatography of Plant Pigments?
Key safety precautions include: Acetone and petroleum ether are highly flammable — no open flames; Work in a well-ventilated area or fume hood; Avoid inhaling solvent vapors.
What materials are needed for Chromatography of Plant Pigments?
The main materials required are: Fresh spinach leaves, Acetone or isopropanol, Petroleum ether, Chromatography paper, Sand.
What results should I expect from Chromatography of Plant Pigments?
Four or more colored bands should be visible. Carotenoids (orange) travel farthest (Rf ≈ 0.9–0.95). Xanthophylls (yellow) at Rf ≈ 0.7. Chlorophyll a (blue-green) at Rf ≈ 0.5. Chlorophyll b (yellow-green) at Rf ≈ 0.4.