Chromatography - TLC, Column, and Paper Chromatography

Real-Life Hook: The Invisible Made Visible

Ever wondered how forensic scientists detect tiny traces of drugs in blood samples? How environmental agencies identify pollutants in water? How your doctor diagnoses diseases from a single drop of blood? The answer is chromatography - literally “color writing”!

From catching criminals (forensic analysis) to ensuring food safety (detecting pesticides) to discovering new drugs (pharmaceutical research), chromatography is the detective’s best friend in chemistry. It can:

• Separate mixtures that look identical to the naked eye
• Identify compounds present in microscopic quantities
• Purify compounds from complex natural sources
• Monitor purity of pharmaceuticals

What started as a technique to separate plant pigments has become one of the most powerful analytical tools in modern science. In fact, several Nobel Prizes have been awarded for advancements in chromatography!

What is Chromatography?

Chromatography is a separation technique based on differential migration of components of a mixture through a stationary phase under the influence of a mobile phase.

Etymology: Greek words “chroma” (color) + “graphein” (to write)

Discovered by: Russian botanist Mikhail Tsvet (1906) while separating plant pigments

The Two Phases

Every chromatographic technique involves:

1. Stationary Phase (doesn’t move)

   • Solid: Silica gel, alumina, paper
   • Liquid: Coated on solid support
   • Provides surface for differential adsorption

2. Mobile Phase (moves)

   • Liquid: Organic solvents
   • Gas: Inert gases (in gas chromatography)
   • Carries components through stationary phase

Basic Principle

Different components of a mixture have different affinities for the stationary and mobile phases:

Classification of Chromatography

Based on Physical State:

1. Liquid Chromatography: Mobile phase is liquid (TLC, column, paper)
2. Gas Chromatography: Mobile phase is gas (GC, GC-MS)

Based on Separation Mechanism:

1. Adsorption Chromatography: TLC, column chromatography
2. Partition Chromatography: Paper chromatography
3. Ion Exchange Chromatography: Separating ions
4. Size Exclusion Chromatography: Separating by molecular size

For JEE: Focus on TLC, Column, and Paper chromatography

Thin Layer Chromatography (TLC)

Principle

TLC is an adsorption chromatography technique where:

• Stationary phase: Thin layer of adsorbent (silica gel or alumina) coated on glass/plastic plate
• Mobile phase: Organic solvent or solvent mixture
• Mechanism: Differential adsorption of components on the stationary phase

Competition: Components compete between:

• Being adsorbed on stationary phase (silica/alumina)
• Being dissolved in mobile phase (solvent)

Why Silica Gel?

Silica gel (SiO₂·xH₂O) is preferred because:

1. Polar surface: Many -OH groups → strong adsorption sites
2. Large surface area: Porous structure → effective separation
3. Inert: Doesn’t react with most compounds
4. Economical: Cheap and readily available

Structure of Silica Gel:

Apparatus and Materials

Materials Required:

1. TLC plate: Glass plate coated with silica gel (0.2-0.5 mm thickness)
2. Capillary tube: For spotting sample
3. Developing chamber: Glass jar with lid (chromatography jar)
4. Mobile phase: Organic solvent(s)
5. Pencil: To mark baseline and solvent front (NEVER pen - ink dissolves!)
6. UV lamp or iodine chamber: For visualization

Detailed Procedure

Step 1: Preparation of TLC Plate

1. Take silica gel-coated glass plate (pre-made or prepare by coating)
2. Draw baseline with pencil (~1.5 cm from bottom)
3. Mark spot positions on baseline (for multiple samples)
4. Activate plate if needed (heat at 110°C for 30 min to remove moisture)

Step 2: Sample Application (Spotting)

1. Dissolve sample in volatile solvent (acetone, methanol)
2. Dip capillary tube in solution
3. Touch capillary lightly to baseline
4. Apply small spot (2-3 mm diameter)
5. Let dry completely
6. Repeat 2-3 times for better visibility (same spot)

⚠️ Critical: Spot should be:

• Small and concentrated (not spread out)
• Well above solvent level in chamber
• Dry before placing in chamber

Step 3: Preparation of Developing Chamber

1. Pour mobile phase in jar (depth: 0.5-1 cm)
2. Add filter paper on inner wall (helps vapor saturation)
3. Close lid and wait 15-20 minutes for saturation
4. Saturated atmosphere ensures uniform solvent movement

Step 4: Development (Running the Chromatogram)

1. Place TLC plate in chamber (spot above solvent!)
2. Close lid immediately
3. Let solvent rise by capillary action
4. When solvent reaches ~1 cm from top, remove plate
5. Immediately mark solvent front with pencil

Interactive Demo: Visualize Chromatography Separation

See how compounds separate based on differential migration in chromatography.

Why mark immediately?: Solvent evaporates quickly, solvent front becomes invisible!

Step 5: Visualization

Method 1: UV Lamp (for UV-active compounds)

• Shine UV light (254 nm or 366 nm)
• Fluorescent compounds appear as bright spots
• Non-fluorescent compounds appear as dark spots (if plate has fluorescent indicator)

Method 2: Iodine Chamber

• Place developed plate in chamber with iodine crystals
• Iodine vapors react with organic compounds
• Brown spots appear
• Mark immediately (spots fade as iodine sublimes)

Method 3: Chemical Sprays

• Ninhydrin: For amino acids (purple spots)
• Sulfuric acid: Chars organic compounds (black spots)
• Specific reagents for specific functional groups

Step 6: Calculation of Rf Value

Mark spot centers and measure:

$$R_f = \frac{\text{Distance traveled by compound}}{\text{Distance traveled by solvent}}$$ $$R_f = \frac{\text{Distance from baseline to spot center}}{\text{Distance from baseline to solvent front}}$$

Properties of Rf:

• Always 0 < Rf < 1
• If Rf = 0 → compound didn’t move (too strongly adsorbed)
• If Rf = 1 → compound moved with solvent front (not adsorbed)
• Ideal Rf = 0.3 to 0.7 (good separation)

Factors Affecting Rf Value

Factor	Effect on Rf	Explanation
Polarity of mobile phase	↑ Polarity → ↑ Rf	Polar solvent competes better with polar stationary phase
Polarity of compound	↑ Polarity → ↓ Rf	Polar compounds adsorb more strongly on polar silica
Temperature	↑ Temp → ↑ Rf	Increases solubility in mobile phase
Humidity	↑ Humidity → ↓ Rf	Water adsorbs on silica, reduces effective adsorption sites
Adsorbent activity	↑ Activity → ↓ Rf	More active adsorbent holds compounds more strongly

Interpretation of TLC Results

Number of Spots:

• 1 spot → Pure compound (or inseparable mixture)
• Multiple spots → Mixture OR degradation products

Position of Spots:

• High Rf (near solvent front) → Non-polar compound
• Low Rf (near baseline) → Polar compound

Comparing Samples:

• Run known standard alongside unknown
• If Rf matches → likely same compound
• Confirm with other techniques (melting point, spectroscopy)

Applications of TLC

1. Monitoring Reaction Progress

   • Take samples at intervals during reaction
   • TLC shows disappearance of starting material
   • Appearance of product spots

2. Checking Purity

   • Pure compound → single spot
   • Multiple spots → impurities present

3. Identifying Compounds

   • Compare Rf with known standards
   • Preliminary identification

4. Optimizing Column Chromatography

   • Test different solvent systems on TLC
   • Choose best system for column separation

5. Pharmaceutical Analysis

   • Checking drug purity
   • Detecting adulterants

Memory Trick: “TLC PREP”

Thin layer of silica (stationary phase) Light spotting (small, concentrated spots) Chamber saturated (with solvent vapors)

Pencil for marking (never pen!) Run chromatogram (solvent rises) Evaluate under UV/iodine Perform Rf calculation

Column Chromatography

Principle

Column chromatography is a preparative technique using adsorption chromatography to separate and purify larger quantities of compounds (grams to kilograms).

Key Difference from TLC:

• TLC: Analytical (milligram scale, identifies components)
• Column: Preparative (gram scale, purifies components)

Apparatus and Setup

Column: Glass tube with:

• Stopcock at bottom (control flow rate)
• Glass wool plug (prevents adsorbent from washing out)
• Fitted at bottom with narrow outlet

Components:

Adsorbents for Column Chromatography

Adsorbent	Polarity	Best For	Particle Size
Silica Gel	Polar	Most organic compounds	60-120 mesh
Alumina (Al₂O₃)	Polar	Alkaline-sensitive compounds	50-200 mesh
Activated Charcoal	Non-polar	Decolorization	Fine powder

Mesh Size: Higher mesh number = smaller particles = better separation (but slower flow)

Types of Alumina

1. Acidic alumina (pH 4): For acidic compounds
2. Neutral alumina (pH 7): For neutral compounds
3. Basic alumina (pH 10): For basic compounds (amines)

Detailed Procedure

Step 1: Packing the Column

1. Dry Method (for expert users):

   • Add dry silica gel to dry column
   • Tap gently to settle uniformly
   • Problem: Can form air pockets

2. Wet Method (recommended):

   • Add solvent to column first
   • Make slurry of silica gel in solvent (separate beaker)
   • Pour slurry into column in one continuous motion
   • Let settle; add more if needed
   • No air bubbles → uniform packing

Column Dimensions:

• Height of adsorbent = 10-20 times the diameter
• Too short → poor separation
• Too tall → excessive spreading (diffusion)

Step 2: Sample Loading

Method 1: Dry Loading

1. Mix sample with small amount of silica gel
2. Evaporate solvent completely (rotary evaporator)
3. Transfer dry sample-silica mixture to top of column
4. Cover with small layer of sand (prevents disturbance)

Method 2: Wet Loading (common)

1. Dissolve sample in minimum amount of solvent
2. Carefully add to top of column with pipette
3. Let it soak into adsorbent
4. Don’t disturb the surface

Critical: Never let column run dry! Always maintain solvent layer above adsorbent.

Step 3: Elution (Running the Column)

1. Add mobile phase solvent to column
2. Open stopcock to control flow rate (1-2 drops/second optimal)
3. Components separate into bands as they move down
4. Less polar compounds move faster (larger Rf)
5. More polar compounds move slower (smaller Rf)

Gradient Elution (for complex mixtures):

• Start with non-polar solvent (hexane)
• Gradually increase polarity (add ethyl acetate)
• Continues to increase polarity (add methanol)
• Each component elutes in order of polarity

Step 4: Collection of Fractions

1. Collect eluent in test tubes (fractions)
2. Each fraction = 5-10 mL
3. Number each fraction
4. Visible colored bands → easy to collect separately
5. Colorless compounds → collect all fractions, analyze by TLC

Step 5: Monitoring Fractions (TLC)

1. Spot each fraction on TLC plate
2. Develop and visualize
3. Fractions with same Rf → same compound → combine
4. Pure fractions → single spot
5. Mixed fractions → re-chromatograph or discard

Step 6: Solvent Removal

1. Combine fractions containing desired compound
2. Evaporate solvent (rotary evaporator)
3. Obtain pure compound
4. Verify purity (m.p., TLC, spectroscopy)

Column Chromatography Variables

1. Adsorbent to Sample Ratio

• Typical: 30:1 to 100:1 (weight ratio)
• More adsorbent → better separation
• Too much → wastage and long time

2. Flow Rate

• Slow (0.5 drop/sec) → better separation, more time
• Fast (2-3 drops/sec) → poor separation, less time
• Optimal: 1-2 drops/second

3. Column Dimensions

• Height/Diameter ratio = 10-20:1
• Longer column → better separation
• Wider column → can load more sample

4. Particle Size

• Smaller particles → better separation, slower flow
• Larger particles → poorer separation, faster flow
• Common: 60-120 mesh

Solvent Systems (Mobile Phase)

Eluotropic Series (increasing polarity):

1. Petroleum ether (least polar)
2. Hexane
3. Cyclohexane
4. Carbon tetrachloride
5. Benzene
6. Chloroform
7. Diethyl ether
8. Ethyl acetate
9. Acetone
10. Methanol
11. Water (most polar)

Selecting Solvent:

• Start with less polar solvent
• Compound should have Rf = 0.3-0.4 on TLC in that solvent
• Gradually increase polarity during elution

Memory Trick - “Please Help Clean Bathrooms Carefully; Don’t Ever Accidentally Mess Walls”

Petroleum ether, Hexane, Cyclohexane, Benzene, Chloroform, Diethyl ether, Ethyl acetate, Acetone, Methanol, Water

Common Mistakes in Column Chromatography

Mistake 1: Column Running Dry

Problem:

• Cracks develop in adsorbent
• Non-uniform flow
• Poor separation
• Sample lost in cracks

Solution: Always maintain solvent level above adsorbent. Add solvent before it completely drains.

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Mistake 2: Air Bubbles in Column

Problem: Disturb uniform flow, cause band spreading

Solution:

• Use wet packing method
• Tap column gently during packing
• If bubble forms, try to dislodge by tapping

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Mistake 3: Disturbing Sample Band

Problem: Sample spreads, poor separation

Solution:

• Add solvent gently (use glass rod or funnel)
• Cover sample with sand layer
• Don’t let solvent fall directly on sample

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Mistake 4: Too Fast Flow Rate

Problem: Components don’t have time to equilibrate between phases

Solution: Control with stopcock - 1-2 drops/second is optimal

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Mistake 5: Overloading Column

Problem:

• Bands overlap
• Poor separation
• Adsorbent saturated

Solution: Use adsorbent:sample ratio of at least 30:1

Applications of Column Chromatography

1. Purification of Natural Products

   • Alkaloids from plants
   • Vitamins from natural sources
   • Pigments (chlorophyll, carotenoids)

2. Purification of Reaction Products

   • Separating product from starting materials
   • Removing byproducts

3. Pharmaceutical Industry

   • Purifying drug molecules
   • Removing impurities

4. Research Laboratories

   • Isolating pure compounds for characterization
   • Preparative scale (grams to kilograms)

Paper Chromatography

Principle

Paper chromatography is a partition chromatography technique:

• Stationary phase: Water molecules trapped in cellulose fibers of paper
• Mobile phase: Organic solvent
• Mechanism: Partition of components between water (stationary) and organic solvent (mobile)

Key Difference from TLC/Column:

• TLC/Column: Adsorption chromatography (surface phenomenon)
• Paper: Partition chromatography (solubility-based)

Why is Water the Stationary Phase?

Paper composition: Cellulose (polysaccharide)

• Contains many -OH groups
• Highly hydrophilic (water-loving)
• Adsorbs water molecules from atmosphere
• This water layer acts as stationary phase

Types of Paper Chromatography

1. Ascending Chromatography (most common)

• Solvent rises up by capillary action
• Paper hangs vertically
• Sample spot near bottom

2. Descending Chromatography

• Solvent descends down by gravity + capillary action
• Paper hangs with solvent trough at top
• Faster than ascending

3. Circular Chromatography

• Paper is circular
• Sample spot at center
• Solvent spreads radially outward
• Components appear as concentric circles

4. Two-Dimensional Chromatography (2D)

• Run chromatography in one direction
• Dry the paper
• Rotate 90° and run again with different solvent
• Superior separation for complex mixtures

Apparatus and Materials

Materials:

1. Chromatography paper: Whatman filter paper (No. 1 or No. 4)
2. Developing chamber: Large glass jar or beaker with cover
3. Mobile phase: Organic solvent mixture
4. Capillary tubes: For spotting
5. Pencil: For markings
6. Clips/hook: To suspend paper

Detailed Procedure (Ascending)

Step 1: Preparation

1. Cut Whatman paper to appropriate size (20 cm × 10 cm)
2. Draw baseline with pencil (2 cm from bottom)
3. Mark spot positions on baseline
4. Handle paper by edges only (oils from fingers interfere!)

Step 2: Sample Application

1. Dissolve sample in volatile solvent
2. Apply small spot using capillary tube
3. Let dry completely
4. Repeat 2-3 times for concentrated spot
5. Spot diameter: 2-3 mm (small!)

Step 3: Chamber Preparation

1. Pour mobile phase in jar (depth: 1 cm)
2. Saturate atmosphere by lining with filter paper soaked in solvent
3. Close and wait 15 minutes

Step 4: Development

1. Suspend paper in chamber (baseline above solvent!)
2. Ensure paper doesn’t touch jar walls
3. Close lid
4. Solvent rises by capillary action
5. Remove when solvent reaches ~1 cm from top
6. Mark solvent front immediately

Step 5: Drying and Visualization

1. Dry paper in air or oven (gentle heat)
2. Visualize spots (same methods as TLC):
   • UV lamp
   • Iodine vapors
   • Ninhydrin spray (amino acids)
   • Specific reagents

Step 6: Calculate Rf

Same as TLC:

$$R_f = \frac{\text{Distance from baseline to spot center}}{\text{Distance from baseline to solvent front}}$$

Common Solvent Systems for Paper Chromatography

Application	Solvent System
Amino acids	n-Butanol : Acetic acid : Water (4:1:1)
Sugars	n-Butanol : Pyridine : Water (6:4:3)
Plant pigments	Petroleum ether : Acetone (9:1)
Phenols	n-Butanol : Ammonia : Water (10:1:10)
General organic compounds	n-Butanol : Ethanol : Water (4:1:2)

Two-Dimensional (2D) Paper Chromatography

Procedure:

1. Apply sample at one corner of square paper (2 cm from each edge)
2. Develop in first solvent (solvent A)
3. Dry completely
4. Rotate paper 90°
5. Develop in second solvent (solvent B) perpendicular to first
6. Components spread in 2D plane
7. Much better separation for complex mixtures!

Application: Separating amino acid mixtures (20 amino acids in proteins)

Comparison: Paper vs TLC

Feature	Paper Chromatography	TLC
Stationary phase	Water in cellulose	Silica gel/alumina
Mechanism	Partition	Adsorption
Time	Slow (hours)	Fast (minutes)
Separation	Good	Excellent
Cost	Very cheap	Moderate
Reproducibility	Lower	Higher
Sensitivity	Lower	Higher
Best for	Hydrophilic compounds (sugars, amino acids)	General organic compounds

Applications of Paper Chromatography

1. Amino Acid Analysis

   • Identifying amino acids in protein hydrolysates
   • Detecting abnormal amino acids in urine (medical diagnosis)

2. Sugar Analysis

   • Separating monosaccharides, disaccharides
   • Food chemistry

3. Plant Pigment Separation

   • Chlorophylls, carotenoids
   • Botany experiments

4. Clinical Chemistry

   • Detecting drugs in biological fluids
   • Identifying metabolites

5. Food Analysis

   • Detecting food additives
   • Identifying natural vs synthetic colors

Advanced Concepts

Retention Factor (Rf) - Deeper Understanding

Why is Rf constant for a compound?

Rf represents the equilibrium distribution of a compound between mobile and stationary phases.

$$R_f = \frac{v_{compound}}{v_{solvent}}$$

Where v = velocity

For adsorption chromatography:

$$R_f = \frac{1}{1 + \frac{k_a \cdot A_s}{V_m}}$$

Where:

• ka = adsorption equilibrium constant
• As = surface area of stationary phase
• Vm = volume of mobile phase

Constant Rf: As long as experimental conditions (temperature, solvent, adsorbent) are same, Rf is constant for a given compound.

Resolution (Rs)

Resolution measures how well two components are separated:

$$R_s = \frac{2(d_2 - d_1)}{w_1 + w_2}$$

Where:

• d1, d2 = distances traveled by compounds 1 and 2
• w1, w2 = widths of spots/bands 1 and 2

Interpretation:

• Rs < 1: Poor separation (spots overlap)
• Rs = 1: Partial separation (just touching)
• Rs > 1.5: Complete separation (baseline resolution)

Separation Efficiency

Theoretical Plates (N) in column chromatography:

$$N = 16 \left(\frac{d}{w}\right)^2$$

Where:

• d = distance traveled by compound
• w = width of band

Higher N = Better separation efficiency

Height Equivalent to Theoretical Plate (HETP):

$$HETP = \frac{L}{N}$$

Where L = column length

Lower HETP = Better column efficiency

Van Deemter Equation

Relates plate height (H = HETP) to flow velocity (u):

$$H = A + \frac{B}{u} + Cu$$

Where:

• A = Eddy diffusion term (multiple flow paths)
• B = Longitudinal diffusion (diffusion along column)
• C = Mass transfer resistance

Implication: There’s an optimal flow rate that gives minimum H (maximum efficiency)!

Practice Problems

Level 1 - JEE Main (Basics)

Problem 1: In TLC, why should the sample spot be above the solvent level in the chamber?

Solution:

If spot is below solvent level:

• Sample dissolves directly into bulk solvent
• No chromatography occurs
• All components mix in solvent
• No separation achieved
• Sample lost!

Correct placement (spot above solvent level):

• Solvent rises by capillary action
• Reaches sample spot gradually
• Components partition between stationary and mobile phases
• Differential migration occurs
• Separation achieved

Analogy: Think of a race starting line. If runners start behind the starting line (in the water), they’re already disqualified!

Answer: Sample spot must be above solvent level so that components are separated by differential migration through the stationary phase, not dissolved directly in bulk solvent.

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Problem 2: Why is pencil used instead of pen for marking in chromatography?

Solution:

Pen ink problems:

• Contains organic dyes/pigments
• Soluble in chromatography solvents
• Will run with the mobile phase
• Creates extra spots on chromatogram
• Interferes with sample analysis
• Can contaminate sample

Pencil advantages:

• Contains graphite (carbon)
• Insoluble in organic solvents
• Doesn’t migrate with mobile phase
• No interference with sample
• Marks remain stationary

Answer: Pencil (graphite) is insoluble in chromatographic solvents and doesn’t interfere with separation, while pen ink dissolves and creates interfering spots.

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Problem 3: A compound shows Rf = 0.8 in TLC. Is this ideal? How can you adjust the Rf to 0.3-0.5 range?

Solution:

Rf = 0.8 Analysis:

• Compound moved 80% of solvent distance
• Weak interaction with stationary phase
• Strong affinity for mobile phase
• Not ideal: Too close to solvent front
• Poor separation from other high-Rf compounds
• Difficult to measure accurately

Ideal Rf = 0.3 to 0.7 (preferably 0.4 to 0.6)

To decrease Rf from 0.8 to 0.3-0.5:

Method 1: Decrease mobile phase polarity

• Use less polar solvent
• Weaker eluting power
• Compound moves slower
• Example: If using 100% ethyl acetate, try 50% ethyl acetate + 50% hexane

Method 2: Increase stationary phase interaction

• Use more polar/active adsorbent
• More aluminum oxide instead of silica
• Fresher silica gel (more active)

Method 3: Lower temperature (less practical)

• Reduces solubility in mobile phase
• Slower migration

Answer: Rf = 0.8 is too high for ideal separation. Decrease mobile phase polarity (use less polar solvent mixture) to bring Rf to 0.3-0.5 range.

Level 2 - JEE Main (Application)

Problem 4: In a TLC experiment, compound A travels 7.2 cm and compound B travels 4.5 cm when the solvent front moves 9.0 cm. Calculate the Rf values. If the same mixture is run in a column of 30 cm length with the same solvent system, estimate how far each compound will travel.

Solution:

Part 1: Calculate Rf values

Given:

• Distance traveled by A (dA) = 7.2 cm
• Distance traveled by B (dB) = 4.5 cm
• Solvent front distance (dsf) = 9.0 cm

$$R_{f(A)} = \frac{d_A}{d_{sf}} = \frac{7.2}{9.0} = 0.8$$ $$R_{f(B)} = \frac{d_B}{d_{sf}} = \frac{4.5}{9.0} = 0.5$$

Part 2: Predict column separation

Assumption: Rf values remain constant under same conditions (same solvent, temperature, adsorbent)

In column of length 30 cm:

For compound A:

$$R_{f(A)} = \frac{d_A}{30}$$ $$0.8 = \frac{d_A}{30}$$ $$d_A = 0.8 \times 30 = 24 \text{ cm}$$

For compound B:

$$R_{f(B)} = \frac{d_B}{30}$$ $$0.5 = \frac{d_B}{30}$$ $$d_B = 0.5 \times 30 = 15 \text{ cm}$$

Answer:

• Rf(A) = 0.8, Rf(B) = 0.5
• In 30 cm column: A travels 24 cm, B travels 15 cm
• Separation: 24 - 15 = 9 cm apart

Practical note: This is idealized. In reality, bands also have width due to diffusion, so actual separation might be less.

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Problem 5: Why is the chromatography chamber saturated with solvent vapors before running TLC?

Solution:

Without saturation:

1. Uneven solvent evaporation:

   • Mobile phase evaporates from plate as it rises
   • Edges dry faster than center
   • Creates irregular solvent front (curved, not straight)
   • Non-reproducible Rf values

2. Concentration effects:

   • Evaporation concentrates solutes at edges
   • Creates “edge effects”
   • Poor separation

3. Variable Rf:

   • Different evaporation rates
   • Changes effective mobile phase composition
   • Rf values not consistent

With vapor saturation:

1. Equilibrium established:

   • Atmosphere saturated with solvent vapor
   • No net evaporation from plate
   • Uniform solvent movement

2. Straight solvent front:

   • Even rise across entire plate width
   • Reproducible Rf values
   • Better separation

3. Consistent results:

   • Can compare Rf values across experiments
   • Reliable identification

How to achieve saturation:

• Line chamber walls with filter paper soaked in mobile phase
• Close chamber 15-20 minutes before use
• Vapors saturate the atmosphere

Answer: Vapor saturation prevents uneven evaporation of mobile phase from the TLC plate, ensuring a straight solvent front, uniform migration, and reproducible Rf values.

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Problem 6: In column chromatography, why must we never let the column run completely dry?

Solution:

Problems if column runs dry:

1. Cracking of adsorbent bed:

   • Adsorbent shrinks when dry
   • Cracks and channels form
   • Non-uniform flow paths created

2. Channeling:

   • Solvent flows through cracks (path of least resistance)
   • Bypasses much of the adsorbent
   • Poor separation efficiency
   • Sample can be lost in cracks

3. Air introduction:

   • Air pockets form in dried regions
   • Disturbs uniform flow
   • Causes band spreading
   • Reduces resolution

4. Difficulty in restarting:

   • Dry adsorbent may not re-wet uniformly
   • Air bubbles trapped
   • May need to repack column

5. Sample loss:

   • Sample trapped in cracks
   • Incomplete elution
   • Low recovery yield

Prevention:

• Always maintain solvent level above adsorbent
• Add fresh solvent before current level reaches adsorbent surface
• Use reservoir or addition funnel for continuous solvent supply
• Close stopcock when adding solvent (prevents air suction)

Answer: Letting column run dry causes adsorbent cracking, channeling, air pockets, and band spreading, leading to poor separation and sample loss. Always maintain solvent level above adsorbent.

Level 3 - JEE Advanced (Conceptual & Numerical)

Problem 7: Explain why paper chromatography is better for separating amino acids than TLC with silica gel, even though TLC generally gives better resolution.

Solution:

Amino Acid Properties:

• Amphoteric (both acidic and basic groups): -NH₃⁺ and -COO⁻
• Highly polar due to zwitterionic form
• Very soluble in water
• Contains multiple hydrogen bonding sites

Why TLC (Silica Gel) is problematic:

1. Too strong interaction:

   • Silica is highly polar (many -OH groups)
   • Amino acids bind very strongly
   • Rf values near zero (don’t move)
   • Poor separation

2. Irreversible adsorption:

   • Ionic interactions between -NH₃⁺ and Si-O⁻
   • Very difficult to elute
   • May require very polar solvents (methanol + NH₃)
   • Even then, tailing and streaking occur

3. Multiple binding modes:

   • Carboxyl group, amino group, and side chains all interact
   • Complex adsorption behavior
   • Poor spot shape

Why Paper Chromatography works well:

1. Partition mechanism:

   • Water (stationary) vs organic solvent (mobile)
   • Amino acids partition based on hydrophilicity
   • Reversible equilibrium (no irreversible binding)

2. Moderate interactions:

   • Hydrogen bonding with water (not too strong)
   • Differential solubility provides separation
   • Good Rf range (0.3-0.7)

3. Suitable polarity range:

   • Water-rich stationary phase accommodates polar amino acids
   • Organic mobile phase (n-butanol/acetic acid) provides differential elution

4. Historical development:

   • Paper chromatography was developed specifically for amino acids
   • Optimized solvent systems available
   • Ninhydrin detection specific for amino acids

Modern solution: Reversed-phase TLC

• Uses C18-modified silica (non-polar stationary phase)
• Water + organic solvent mobile phase
• Works well for amino acids

Answer: Amino acids are too polar for normal-phase TLC (silica gel), showing very low Rf values and poor separation due to strong ionic interactions. Paper chromatography uses partition between water and organic solvent, providing moderate and differential retention that separates amino acids effectively. The partition mechanism is better suited for highly polar, zwitterionic compounds than adsorption on polar silica.

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Problem 8: In a column chromatography separation, the column is 40 cm long and contains 80 g of silica gel. A mixture of 2.0 g containing compounds A and B is loaded. After running with hexane:ethyl acetate (80:20), compound A elutes in fractions 5-8 (200 mL total) and compound B in fractions 15-20 (300 mL total). Calculate: (a) The adsorbent:sample ratio (b) If each fraction is 50 mL, estimate the Rf values of A and B (c) The separation achieved between A and B

Solution:

Given:

• Column length = 40 cm
• Silica gel = 80 g
• Sample = 2.0 g (A + B)
• Compound A: Fractions 5-8 (4 fractions × 50 mL = 200 mL)
• Compound B: Fractions 15-20 (6 fractions × 50 mL = 300 mL)

(a) Adsorbent:Sample Ratio

$$\text{Ratio} = \frac{\text{Mass of silica gel}}{\text{Mass of sample}} = \frac{80 \text{ g}}{2.0 \text{ g}} = \frac{40}{1}$$

Answer (a): Adsorbent:sample ratio = 40:1

Interpretation: This is a good ratio (typical range 30:1 to 100:1). Sufficient adsorbent for effective separation.

(b) Estimate Rf Values

Approach: In column chromatography, Rf relates to the fraction number and volume eluted.

For compound A:

• Elutes in fractions 5-8
• Mean fraction = (5+8)/2 = 6.5
• Volume eluted = 6.5 × 50 mL = 325 mL

For compound B:

• Elutes in fractions 15-20
• Mean fraction = (15+20)/2 = 17.5
• Volume eluted = 17.5 × 50 mL = 875 mL

Approximation for Rf: In column chromatography, Rf is related to elution volume, but exact calculation requires void volume and column volume.

Simplified estimate:

$$R_f \approx \frac{V_0}{V_e}$$

Where V0 = void volume (space between particles) and Ve = elution volume

Assuming void volume ≈ 0.4 × column volume (typical for packed columns):

If we take total volumes eluted as proxy:

• Compound with smaller elution volume → higher Rf (less retained)
• Compound with larger elution volume → lower Rf (more retained)

Relative Rf:

$$\frac{R_{f(A)}}{R_{f(B)}} \approx \frac{V_{e(B)}}{V_{e(A)}} = \frac{875}{325} \approx 2.7$$

This means A is about 2.7 times more mobile than B.

If we assume a reasonable range:

• Compound A (less retained): Rf ≈ 0.6
• Compound B (more retained): Rf ≈ 0.6/2.7 ≈ 0.22

Answer (b): Estimated Rf(A) ≈ 0.5-0.6, Rf(B) ≈ 0.2-0.3

(c) Separation Between A and B

Method 1: Fraction difference

• A: Fractions 5-8
• B: Fractions 15-20
• Gap: Fractions 9-14 (no overlap!)
• Excellent baseline separation

Method 2: Volume difference

• End of A elution: Fraction 8 = 400 mL
• Start of B elution: Fraction 15 = 750 mL
• Gap: 350 mL with no compound elution

Method 3: Resolution For baseline resolution, Rs > 1.5

Given no overlap of fractions, Rs » 1.5 (complete separation)

Answer (c): Complete baseline separation achieved. No overlap between fractions containing A and B, indicating excellent chromatographic separation.

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Problem 9: A student performs TLC on a mixture and observes one spot with Rf = 0.6. On injecting the same mixture into a GC-MS instrument, three peaks appear. Explain this observation and suggest how the student could modify the TLC experiment to detect all three components.

Solution:

Analysis of Discrepancy:

TLC shows: 1 spot (appears pure) GC-MS shows: 3 peaks (actually a mixture of 3 compounds)

Possible Reasons:

1. Similar Rf values:

   • All three compounds have nearly identical Rf in current solvent system
   • Spots overlap completely
   • Appear as single spot

2. Poor resolution:

   • Components not sufficiently separated in current conditions
   • Spots merge into one

3. Detection limitation:

   • One or two components might be present in very small amounts
   • Below detection limit of visualization method
   • GC-MS is much more sensitive than TLC visualization

4. Similar polarity:

   • Three compounds might be structural isomers
   • Or have similar functional groups
   • Similar chromatographic behavior

How to Modify TLC to Detect All Three:

Method 1: Change Mobile Phase (Most Important)

• Try different solvent systems
• Test solvents of different polarities
• Use mixed solvent systems in different ratios
• Example: If using ethyl acetate:hexane (1:1), try:
  • 30:70 (less polar)
  • 70:30 (more polar)
  • Or completely different system like chloroform:methanol

Method 2: Use 2D-TLC

• Run TLC in one direction with solvent A
• Dry the plate
• Run perpendicular with solvent B
• Often separates compounds with similar Rf in one dimension

Method 3: Change Stationary Phase

• Instead of silica gel, try:
  • Aluminum oxide (alumina)
  • Reversed-phase TLC (C18)
  • Different silica gel particle size/activity

Method 4: Improve Detection

• Try different visualization methods:
  • Multiple UV wavelengths (254 nm and 366 nm)
  • Different chemical sprays
  • Iodine vapors
• One component might be UV-inactive but visible with iodine

Method 5: Develop Twice

• Run TLC, dry, run again in same direction with same solvent
• Multiple developments increase separation
• Each development is like adding theoretical plates

Method 6: Use Better Quality TLC Plates

• HPTLC (High Performance TLC) plates
• Smaller particle size
• Better resolution
• Can separate compounds with Rf differences as small as 0.05

Systematic Approach:

1. First: Change mobile phase polarity (easiest and most effective)
2. If still one spot: Try completely different solvent system
3. If still one spot: Try 2D-TLC or different stationary phase
4. If still one spot: Might be isomers that only GC can separate (temperature-dependent separation)

Answer: The three compounds likely have very similar Rf values in the current TLC system, causing spots to overlap and appear as one. To detect all three:

1. Change mobile phase to different polarity/composition
2. Try 2D-TLC with two different solvent systems
3. Use different stationary phase (alumina or reversed-phase)
4. Try multiple development or HPTLC for better resolution
5. Improve detection methods (different UV wavelengths, chemical sprays)

The most effective approach is systematic variation of mobile phase polarity until separation is achieved.

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Problem 10: Derive the relationship between Rf and the partition coefficient (K) for paper chromatography. If a compound has partition coefficient K = 3 (favoring mobile phase), calculate its Rf value.

Solution:

Partition Coefficient Definition:

For paper chromatography (partition between stationary aqueous phase and mobile organic phase):

$$K = \frac{[\text{Compound in mobile phase}]}{[\text{Compound in stationary phase}]}$$

Derivation:

Let:

• Cs = concentration in stationary phase
• Cm = concentration in mobile phase
• Vs = volume of stationary phase
• Vm = volume of mobile phase
• vs = velocity of compound
• vm = velocity of mobile phase (solvent front)

Time spent in each phase:

Total time for solvent to travel distance d:

$$t = \frac{d}{v_m}$$

Time compound spends in mobile phase:

$$t_m = \frac{d_c}{v_m}$$

where dc = distance traveled by compound

Fraction of time in mobile phase:

$$f_m = \frac{C_m \cdot V_m}{C_m \cdot V_m + C_s \cdot V_s}$$

Since compound only moves when in mobile phase:

$$R_f = \frac{d_c}{d} = f_m$$

Substituting K:

$$K = \frac{C_m}{C_s}$$

Therefore:

$$C_m = K \cdot C_s$$ $$R_f = \frac{K \cdot C_s \cdot V_m}{K \cdot C_s \cdot V_m + C_s \cdot V_s}$$ $$R_f = \frac{K \cdot V_m}{K \cdot V_m + V_s}$$

Dividing numerator and denominator by Vm:

$$R_f = \frac{K}{K + \frac{V_s}{V_m}}$$

Let phase ratio $\beta = \frac{V_s}{V_m}$ (typically ~0.2-0.5 for paper chromatography)

$$\boxed{R_f = \frac{K}{K + \beta}}$$

Or alternatively:

$$\boxed{K = \frac{R_f \cdot \beta}{1 - R_f}}$$

Calculation for K = 3:

Assuming typical phase ratio β ≈ 0.3 for paper chromatography:

$$R_f = \frac{K}{K + \beta} = \frac{3}{3 + 0.3} = \frac{3}{3.3} = 0.909$$

Verification: Higher K means compound favors mobile phase → moves faster → higher Rf ✓

Answer:

• Derived relationship: $R_f = \frac{K}{K + \beta}$ where β is the phase ratio (Vs/Vm)
• For K = 3 and typical β = 0.3: Rf ≈ 0.91

Physical Interpretation:

• K = 3: Compound prefers mobile phase by 3:1 ratio
• Spends 3/(3+0.3) ≈ 90.9% of time in mobile phase
• Therefore travels 90.9% of solvent distance
• Rf = 0.909

Special Cases:

• K → 0 (highly polar, stays in stationary phase): Rf → 0
• K → ∞ (non-polar, stays in mobile phase): Rf → 1
• K = β (equal preference): Rf = 0.5

Cross-Links to Related Topics

Organic Chemistry Connections

1. Crystallization: Complementary purification - chromatography for liquids/mixtures, crystallization for solids

2. Distillation: Compare separation principles - chromatography (polarity/adsorption) vs distillation (volatility)

3. Detection of Elements: After chromatographic purification, detect elements in pure compound

4. Organic Preparations: Chromatography used to purify and analyze organic synthesis products

5. Organic Tests: TLC can identify functional groups by Rf patterns

6. Hydrocarbons: Separating aromatic vs aliphatic hydrocarbons

Physical Chemistry Connections

1. Solutions: Partition coefficients, solubility, intermolecular forces

2. Adsorption: Surface chemistry, adsorption isotherms (Langmuir, Freundlich)

3. Chemical Equilibrium: Partition equilibrium between phases

4. Thermodynamics: Gibbs free energy of adsorption, temperature effects

Practical Chemistry Connections

1. Qualitative Analysis: Paper chromatography for identifying ions

2. Volumetric Analysis: Assessing purity of compounds after chromatographic separation

Analytical Applications

1. Biomolecules: Separating amino acids, sugars, proteins

2. Natural Products: Isolating compounds from plant extracts

Key Takeaways

1. Chromatography principle: Differential migration based on partition/adsorption between stationary and mobile phases

2. TLC: Fast, analytical, adsorption-based; uses silica gel/alumina; minutes

3. Column Chromatography: Slow, preparative, same principle as TLC; uses larger amounts; grams scale

4. Paper Chromatography: Partition-based; cellulose + water vs organic solvent; best for polar compounds (amino acids, sugars)

5. Rf value: Distance traveled by compound / Distance traveled by solvent; characteristic for each compound under specific conditions

6. Ideal Rf: 0.3-0.7 for good separation and measurement

7. Visualization: UV lamp, iodine vapors, chemical sprays (ninhydrin for amino acids)

8. Critical factors: Stationary phase activity, mobile phase polarity, temperature, humidity

9. Column tips: Never let dry, control flow rate (1-2 drops/sec), use 30:1 adsorbent:sample ratio

10. Applications: Monitoring reactions, checking purity, identifying compounds, purifying natural products

Quick Revision Points

✓ TLC: Analytical, silica/alumina, adsorption, fast (30 min), Rf = 0.3-0.7 ideal ✓ Column: Preparative, same stationary phase as TLC, slower, never run dry ✓ Paper: Partition, water in cellulose, best for polar/hydrophilic compounds ✓ Rf formula: distance of spot / distance of solvent front ✓ Spotting: Small, concentrated, above solvent level, pencil markings only ✓ Visualization: UV (254/366 nm), iodine, ninhydrin (amino acids) ✓ Polarity series: Hexane < CHCl₃ < EtOAc < Acetone < MeOH < Water ✓ Polar compound: Low Rf (strong adsorption); Non-polar: High Rf (weak adsorption) ✓ 2D chromatography: Two solvents, 90° rotation, complex mixture separation ✓ Applications: Purity check, reaction monitoring, compound identification, preparative purification

Master chromatography, and you hold the key to separating the inseparable!

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Interactive Demo: Visualize Chromatographic Separation

Watch how compounds migrate through stationary and mobile phases with different affinities.