Separation of pigments from leaf/flower extracts via paper chromatography

A Comprehensive Laboratory Experiment Guide

Paper chromatography is a fundamental analytical technique used to separate and identify pigments present in plant materials. This experiment demonstrates the separation of chlorophyll and other pigments from leaf and flower extracts, allowing students to understand the principles of chromatography and calculate Rf (retention factor) values.

Aim

To separate pigments from leaf/flower extracts using paper chromatography and determine their Rf values for identification purposes.

Apparatus Required

• Whatman filter paper (chromatography paper)
• Test tubes (large and small)
• Capillary tubes
• Beakers (100 mL)
• Glass rod
• Pencil and ruler
• Scalpel or sharp blade
• Solvent system (petroleum ether and acetone mixture)
• Fresh green leaves (spinach, mint) and colored flowers
• Mortar and pestle
• Sand powder
• Filter paper
• Cork or rubber stopper
• Measuring cylinder

Theory

Principle of Paper Chromatography

Paper chromatography is based on the differential partitioning of compounds between a stationary phase (cellulose fibers of filter paper) and a mobile phase (solvent mixture). The separation occurs due to varying affinities of pigments for the two phases.

Pigments in Plants

Plant tissues contain several pigments:

• Chlorophyll a: Blue-green pigment, primary photosynthetic pigment
• Chlorophyll b: Yellow-green pigment, accessory photosynthetic pigment
• Carotenoids: Yellow to orange pigments (carotenes and xanthophylls)
• Anthocyanins: Red, purple, or blue pigments found in flowers and fruits

Rf Value Calculation

The retention factor (Rf) is a characteristic value for each compound under specific conditions:

Rf = Distance traveled by compound / Distance traveled by solvent front

Solvent System

Petroleum ether and acetone mixture (9:1 ratio) is commonly used because:

• Petroleum ether provides non-polar environment
• Acetone increases polarity slightly
• Allows separation of pigments based on their polarity

Procedure

Preparation

1. Sample Preparation:

   • Collect fresh green leaves and colored petals
   • Crush 2-3 grams of material with sand and a few drops of acetone in a mortar
   • Grind thoroughly to extract pigments
   • Filter the extract through filter paper into a clean test tube

2. Chromatography Setup:

   • Cut chromatography paper to appropriate size (20 cm long, 2 cm wide)
   • Draw a straight line 2 cm from the bottom using pencil (origin line)
   • Make a small spot of extract on the origin line using capillary tube
   • Allow the spot to dry completely and repeat 3-4 times

3. Solvent Preparation:

   • Prepare petroleum ether:acetone mixture in 9:1 ratio
   • Pour solvent to a depth of 1 cm in a large test tube

Chromatography Process

1. Loading:

   • Place the prepared paper strip in the test tube
   • Ensure the spot remains above the solvent level
   • Cover the test tube with a stopper

2. Development:

   • Allow solvent to rise up the paper by capillary action
   • Remove the paper when solvent front reaches 15-18 cm
   • Mark the solvent front immediately with pencil

3. Analysis:

   • Mark the position of each separated pigment band
   • Measure distances from origin line to center of each band
   • Calculate Rf values for each pigment

Observation Table

Note: Values may vary based on experimental conditions and sample type

Result

The paper chromatography successfully separated different pigments from leaf/flower extracts. The following observations were made:

1. Separation Achieved: Four distinct pigments were clearly separated
2. Rf Values: Each pigment showed characteristic Rf values
3. Pigment Identification:
   • Chlorophyll b (Rf ≈ 0.15-0.20)
   • Chlorophyll a (Rf ≈ 0.20-0.25)
   • Xanthophylls (Rf ≈ 0.40-0.50)
   • Carotenes (Rf ≈ 0.50-0.70)

The experiment validates that different pigments have varying solubilities in the solvent system and different affinities for the stationary phase, resulting in distinct separation patterns.

Precautions

1. Handle solvents carefully:

   • Work in well-ventilated area
   • Avoid skin contact with petroleum ether and acetone
   • Keep solvents away from open flames

2. Maintain experimental conditions:

   • Keep temperature constant throughout the experiment
   • Ensure uniform strip dimensions
   • Use fresh solvent for each experiment

3. Proper technique:

   • Apply samples sparingly to avoid spreading
   • Keep the developing chamber covered
   • Mark solvent front immediately after development

4. Sample preparation:

   • Use fresh plant material for better pigment extraction
   • Ensure complete grinding of samples
   • Filter extracts properly to remove debris

5. Measurement accuracy:

   • Use sharp pencil for markings
   • Measure distances carefully from the same reference point
   • Record observations immediately

Viva Voce Questions and Answers

Q1: What is the principle behind paper chromatography?

A: Paper chromatography is based on the differential partitioning of compounds between a stationary phase (cellulose fibers in filter paper) and a mobile phase (solvent mixture). Compounds with higher affinity for the mobile phase travel faster than those with higher affinity for the stationary phase.

Q2: Why is petroleum ether used in the solvent system?

A: Petroleum ether is a non-polar solvent that helps dissolve non-polar pigments like carotenes. Its low polarity allows proper separation of pigments based on their differing polarities.

Q3: What does Rf value indicate?

A: Rf (retention factor) value indicates how far a compound travels relative to the solvent front. It is characteristic for each compound under specific experimental conditions and helps in identification.

Q4: Why are pencil marks used instead of ink?

A: Pencil marks (made of graphite) are inert and don't dissolve in the solvent, unlike ink which may spread or interfere with the separation process.

Q5: What is the significance of repeating the spotting process?

A: Repeating the spotting process concentrates the sample, making the pigment bands more visible and distinct for better analysis and accurate Rf value determination.

Q6: How does the polarity of pigments affect their Rf values?

A: More polar pigments have lower Rf values because they have higher affinity for the polar stationary phase (cellulose) and move slower. Non-polar pigments have higher Rf values as they prefer the mobile phase.

Q7: What safety measures should be taken while handling the solvents?

A: Work in a well-ventilated area, avoid skin contact, keep away from flames, and properly dispose of used solvents according to laboratory guidelines.

Q8: Why is sand added during sample preparation?

A: Sand acts as an abrasive agent that helps in grinding the plant material more effectively, facilitating better extraction of pigments from the cells.

Applications and Significance

This experiment has several practical applications:

1. Educational Value:

   • Teaches fundamental chromatography principles
   • Develops laboratory skills and observation techniques
   • Demonstrates analytical chemistry concepts

2. Research Applications:

   • Pigment analysis in plant studies
   • Quality control in food industry
   • Environmental monitoring

3. Industrial Applications:

   • Natural colorant production
   • Pharmaceutical research
   • Cosmetic industry

Conclusion

The separation of pigments from plant extracts using paper chromatography is a classic experiment that effectively demonstrates chromatographic principles. The determination of Rf values provides quantitative data for pigment identification and comparison. This technique remains valuable for both educational purposes and practical applications in various scientific fields.

The experiment successfully validates the differential mobility of pigments in a solvent system, with each compound exhibiting characteristic Rf values. Proper execution of the procedure, attention to experimental conditions, and careful measurement techniques ensure reliable and reproducible results.