๐ 1. Introduction to Chromatography
Chromatography is a powerful analytical technique used for the separation, identification, and purification of components in a mixture.
Etymology: The term "chromatography" comes from Greek words "chroma" (color) and "graphein" (to write), literally meaning "color writing."
- Discovered by: Russian botanist Mikhail Tswett in 1906 while separating plant pigments
- Principle: Based on differential distribution of mixture components between two phases
- Nature: Physical separation method (no chemical change occurs)
Key Insight: Chromatography exploits the different affinities of substances for two phases - one moving, one stationary.
โ๏ธ 2. Principle of Chromatography
Two-Phase System
STATIONARY PHASE
Fixed in place
Examples: Paper, Silica gel, Alumina
Fixed in place
Examples: Paper, Silica gel, Alumina
โท
MOBILE PHASE
Moves through stationary phase
Examples: Solvent, Gas
Moves through stationary phase
Examples: Solvent, Gas
๐ฌ Separation Mechanisms:
- Adsorption: Components adhere to stationary phase surface with different strengths
- Partition: Components distribute between two liquid phases
- Size Exclusion: Separation based on molecular size
- Ion Exchange: Separation based on ionic charge
Real-life Analogy: Think of chromatography like a race where different runners (components) run at different speeds through obstacles (stationary phase), causing them to separate over time.
๐งช 3. Types of Chromatography
๐ Paper Chromatography
- Stationary Phase: Cellulose paper (water molecules trapped in cellulose fibers)
- Mobile Phase: Solvent (water or organic solvent)
- Mechanism: Partition chromatography
- Applications: Separating plant pigments, amino acids, sugars
Paper Chromatography Setup
1. Paper strip with sample spot near bottom
2. Solvent reservoir at bottom
3. Closed chamber to prevent evaporation
4. Solvent rises by capillary action
5. Components separate as they travel up
2. Solvent reservoir at bottom
3. Closed chamber to prevent evaporation
4. Solvent rises by capillary action
5. Components separate as they travel up
Practical Tip: Use a pencil (not pen) to mark the solvent front and origin line - ink will interfere with results!
๐ฌ Thin Layer Chromatography (TLC)
- Stationary Phase: Thin layer of silica gel or alumina on glass/plastic plate
- Mobile Phase: Organic solvent
- Mechanism: Adsorption chromatography
- Advantages: Faster, better resolution, smaller sample size needed
Safety Note: TLC often uses organic solvents - work in well-ventilated area and avoid inhalation.
๐๏ธ Column Chromatography
- Stationary Phase: Adsorbent packed in a column (alumina, silica gel)
- Mobile Phase: Solvent system
- Purpose: Preparative separation and purification
- Scale: Can handle larger quantities compared to paper/TLC
Industrial Example: Purification of pharmaceutical compounds, isolation of natural products from plant extracts.
๐จ Gas Chromatography (GC)
- Mobile Phase: Inert carrier gas (Helium, Nitrogen)
- Stationary Phase: Non-volatile liquid coated on solid support
- Sample Requirements: Volatile or can be made volatile
- Detection: Highly sensitive detectors (FID, MS)
Modern Applications: Forensic analysis, environmental monitoring, food safety testing, drug screening.
๐ Comparison of Chromatography Types
| Type | Speed | Resolution | Sample Size | Cost | Best Use |
|---|---|---|---|---|---|
| Paper | Slow | Low | Small | Very Low | Educational, qualitative |
| TLC | Fast | Medium | Very Small | Low | Monitoring reactions |
| Column | Medium | High | Large | Medium | Purification |
| Gas | Very Fast | Very High | Very Small | High | Precise analysis |
๐ 4. Retention Factor (Rf) Value
Rf = Distance moved by solute / Distance moved by solvent front
๐ข Key Properties of Rf:
- Range: Always between 0 and 1 (0 < Rf < 1)
- Units: Dimensionless (ratio of distances)
- Specificity: Characteristic for each compound under specific conditions
- Factors affecting Rf: Solvent system, temperature, humidity, stationary phase
๐งฎ Practice Problems
Problem 1: In a paper chromatography experiment:
Rf(A) = 8.4/12 = 0.70
Rf(B) = 4.8/12 = 0.40
- Solvent front moved: 12 cm
- Compound A moved: 8.4 cm
- Compound B moved: 4.8 cm
Rf(A) = 8.4/12 = 0.70
Rf(B) = 4.8/12 = 0.40
Problem 2: If a dye has Rf = 0.65 and solvent front is at 9 cm, how far did the dye travel?
Solution: Distance = Rf ร Solvent front = 0.65 ร 9 = 5.85 cm
Solution: Distance = Rf ร Solvent front = 0.65 ร 9 = 5.85 cm
Interpretation: Higher Rf means greater affinity for mobile phase (more soluble), lower Rf means greater affinity for stationary phase (more strongly adsorbed).
โ๏ธ 5. Factors Affecting Chromatographic Separation
๐ก๏ธ Environmental Factors:
- Temperature: Higher temperature increases molecular motion, affects Rf values
- Humidity: Affects paper chromatography especially
- Pressure: Important in gas chromatography
๐งช Chemical Factors:
- Solvent polarity: Polar solvents move polar compounds better
- pH: Affects ionization of compounds
- Stationary phase: Particle size, surface area, chemical nature
Solvent Selection Guide:
โข Non-polar compounds: Use non-polar solvents (hexane, benzene)
โข Polar compounds: Use polar solvents (water, methanol)
โข Mixed systems: Use solvent mixtures for complex separations
โข Non-polar compounds: Use non-polar solvents (hexane, benzene)
โข Polar compounds: Use polar solvents (water, methanol)
โข Mixed systems: Use solvent mixtures for complex separations
๐ฏ 6. Applications of Chromatography
๐ฌ Scientific Applications:
- Quality Control: Checking purity of pharmaceuticals
- Environmental Analysis: Detecting pollutants in water/air
- Food Industry: Detecting additives, preservatives, contaminants
- Forensics: Drug testing, ink analysis, DNA fingerprinting
๐ฅ Medical Applications:
- Drug Monitoring: Therapeutic drug monitoring in blood
- Metabolomics: Studying metabolic disorders
- Hormone Analysis: Detecting hormone levels
Case Study: Forensic ink analysis can determine if a document was written with the same pen by comparing the Rf values of different ink components.
๐ก 7. Practical Tips and Common Mistakes
โ Best Practices:
- Sample Application: Keep spots small and concentrated
- Solvent Level: Should be below the sample spot initially
- Chamber Saturation: Allow chamber to equilibrate with solvent vapor
- Documentation: Mark solvent front immediately after removal
โ Common Mistakes to Avoid:
Mistake 1: Using pen instead of pencil - ink will run and interfere
Mistake 2: Overloading sample - causes tailing and poor separation
Mistake 3: Not allowing chamber to equilibrate - leads to poor reproducibility
Mistake 4: Disturbing the setup during development - causes uneven solvent flow
๐ 8. Chapter Summary
๐ฏ Key Takeaways:
- Chromatography is a physical separation technique based on differential distribution
- All chromatography involves a stationary phase and a mobile phase
- Rf values are characteristic constants used for compound identification
- Different types serve different purposes: analytical vs. preparative
- Proper technique is crucial for reproducible results
Remember the Formula:
Rf = Distance moved by solute / Distance moved by solvent front
Rf = Distance moved by solute / Distance moved by solvent front
๐ 9. Quick Review Questions
Test Your Knowledge:
- What is the principle behind chromatographic separation?
- Why is the Rf value always less than 1?
- Which type of chromatography would you use to separate volatile compounds?
- What factors affect the Rf value of a compound?
- How does temperature affect chromatographic separation?
Study Strategy: Practice calculating Rf values and try to predict which compounds will have higher/lower Rf values based on their polarity.
๐ 10. Multiple Choice Questions (MCQs)
๐ฏ Single Correct Option Questions:
Q1. The term 'chromatography' was coined by:
Q2. In paper chromatography, the stationary phase is:
Q3. If a compound moves 6 cm and the solvent front moves 10 cm, the Rf value is:
Q4. Which chromatography technique is fastest?
Q5. The Rf value of a compound is always:
Q6. In TLC, the stationary phase is usually:
Q7. Which separation mechanism is NOT involved in chromatography?
Q8. In gas chromatography, the mobile phase is:
Q9. A compound with higher Rf value has:
Q10. Column chromatography is primarily used for: