🎯 Objective
To separate and identify the pigments present in a given plant extract (spinach/grass) by ascending paper chromatography and calculate Rf values.
📖 Principle / Theory
Paper chromatography is an analytical technique where components in a mixture are separated based on their differential partitioning between a stationary phase (cellulose paper) and a mobile phase (solvent). Each component moves with the solvent front at a characteristic rate, expressed as the Retention Factor (Rf):
Rf = Distance travelled by component / Distance travelled by solvent front
Plant pigments separated (in order of increasing Rf): Chlorophyll b → Chlorophyll a → Xanthophylls → Carotenes
🧰 Apparatus Required
Chromatography paper (Whatman No. 1), chromatography tank/jar, capillary tubes, ruler, pencil, UV lamp.
🧪 Chemicals Required
Spinach/grass leaves, petroleum ether, acetone (80:20), chromatography solvent (hexane:acetone 7:3).
⚗️ Procedure
- Cut a strip of Whatman No. 1 chromatography paper (20 cm × 3 cm). Draw a pencil baseline 2 cm from the bottom.
- Grind fresh spinach/grass leaves with acetone. Filter and concentrate the extract.
- Apply small spots of the extract on the baseline using a capillary tube. Allow to dry and apply 5–6 times on the same spot.
- Pour solvent (petroleum ether:acetone, 7:3) to a depth of 1 cm in the chromatography jar. Allow the vapour to saturate the chamber.
- Place the chromatography strip in the jar (spot end down) without touching the wall. Cover the jar.
- Allow the solvent to rise until 1 cm from the top. Remove the strip and immediately mark the solvent front with pencil.
- Allow to dry. Observe the coloured bands (or under UV light for better visibility).
- Measure the distance travelled by each band from the baseline. Calculate Rf values.
📊 Observations & Calculations
| Pigment Band | Colour | Distance from baseline (cm) | Solvent front (cm) | Rf value |
|---|---|---|---|---|
| Band 1 (top) | Orange-yellow | ______ | ______ | ______ |
| Band 2 | Yellow | ______ | ______ | |
| Band 3 | Blue-green | ______ | ______ | |
| Band 4 (bottom) | Yellow-green | ______ | ______ | |
| Solvent front distance | ______ cm | |||
Rf = Distance travelled by pigment / Distance travelled by solvent front
Expected: Carotene ≈ 0.90 | Xanthophyll ≈ 0.65 | Chlorophyll a ≈ 0.40 | Chlorophyll b ≈ 0.20
✅ Result
The plant extract shows ______ bands. The Rf values are: Band 1 = _____, Band 2 = _____, Band 3 = _____, Band 4 = _____. The pigments identified are Carotene, Xanthophyll, Chlorophyll a, and Chlorophyll b.
⚠️ Precautions
- Do not touch the paper with bare hands — oils from skin contaminate the stationary phase.
- The pencil line (baseline) must be light and thin.
- Spots should be small (2–3 mm diameter) and concentrated.
- Ensure the solvent level is below the baseline; paper must not be immersed in solvent.
- Mark the solvent front immediately when the paper is removed.
❓ Viva-Voce Questions
📚 References
- Skoog, D. A. – Principles of Instrumental Analysis
- Vogel's Textbook of Quantitative Chemical Analysis