Paper Chromatography

Chlorophyll Separation

Preparation of Solvent 

✔ Recommended Solvent Mixture (Standard)

A widely used solvent mixture is:

Petroleum ether : Acetone = 9 : 1 (v/v)

👉 This is ideal for separating:

Carotene (orange)

Xanthophyll (yellow)

Chlorophyll a (blue-green)

Chlorophyll b (yellow-green)

🧪 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:

👉 “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.

🔬 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)

🎯 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.

🌿 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

📏 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.

📊 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

👉 Note: Values may vary depending on solvent system and conditions.

🔍 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:

👉 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.