Chlorophyll Separation
Preparation of Solvent
✔ Recommended Solvent Mixture (Standard)
A widely used solvent mixture is:
Petroleum ether : Acetone = 9 : 1 (v/v)
đ This is ideal for separating:
Carotene (orange)
Xanthophyll (yellow)
Chlorophyll a (blue-green)
Chlorophyll b (yellow-green)
đ§Ē Preparation of Solvent (Step-by-Step)
Materials Required:
Petroleum ether (boiling range 40–60°C)
Acetone (analytical grade)
Measuring cylinder
Stoppered bottle / reagent bottle
Procedure:
1. Take a clean, dry measuring cylinder.
2. Measure 90 ml of petroleum ether.
3. Add 10 ml of acetone slowly.
4. Transfer the mixture into a stoppered bottle.
5. Close tightly and mix gently by shaking.
6. Label the bottle:
đ “Chromatography solvent: Petroleum ether : Acetone (9:1)”
⚠️ Important Precautions
Prepare solvent in a well-ventilated area (both solvents are volatile).
Keep away from open flames (highly flammable).
Use fresh solvent for better separation.
Ensure airtight container to prevent evaporation.
đŦ Alternative Solvent Systems (Optional)
You can also use:
1. Petroleum ether : Ethanol (9:1)
2. Hexane : Acetone (8:2)
3. Benzene : Acetone (9:1) (less preferred due to toxicity)
đ¯ Role of Solvent
The solvent (mobile phase) carries pigments upward.
Separation occurs based on polarity differences:
Carotene (non-polar) → moves fastest
Chlorophyll b (more polar) → moves slowest
Identification of pigments
To identify chlorophyll pigments after they move on the stationary phase (paper or TLC), you rely on a combination of colour, position (distance moved), and Rf value.
đŋ Typical Separation Pattern of Plant Pigments
After development, you will observe distinct coloured bands arranged from top to bottom:
Pigment Colour Relative Position Polarity Movement
Carotene Orange Top (farthest) Non-polar Fastest
Xanthophyll Yellow Below carotene Slightly polar Fast
Chlorophyll a Blue-green Middle Moderately polar Medium
Chlorophyll b Yellow-green Bottom (near origin) Most polar Slowest
đ Identification Using Rf Value
The Rf (Retention factor) helps confirm pigment identity:
Rf = \frac{\text{Distance travelled by pigment}}{\text{Distance travelled by solvent front}}
✔ Steps to Calculate Rf:
1. Measure distance from origin to pigment band (cm).
2. Measure distance from origin to solvent front (cm).
3. Apply the formula.
đ Typical Rf Values (approximate)
Pigment Rf Value (approx.)
Carotene 0.90 – 0.95
Xanthophyll 0.70 – 0.80
Chlorophyll a 0.50 – 0.60
Chlorophyll b 0.30 – 0.45
đ Note: Values may vary depending on solvent system and conditions.
đ Key Principles Behind Identification
Polarity rule:
Non-polar pigments travel farther with non-polar solvent.
Polar pigments stick more to polar stationary phase (paper/silica).
Colour recognition:
Each pigment has a characteristic colour.
Order of movement:
đ Top → Bottom:
Carotene → Xanthophyll → Chlorophyll a → Chlorophyll b
⚠️ Precautions for Accurate Identification
Mark the solvent front immediately after removal.
Do not disturb the bands while drying.
Use fresh extract for clear separation.
Avoid overloading the sample spot.