Column Chromatography & Fluorene Oxidation – Detailed Study Notes

Experiment Context

• Last technique experiment in Introductory Organic Lab; FIRST full-scale synthetic experiment
  • Reports (PreLab, InLab, PostLab) now longer and more detailed
• Goal: oxidize fluorene → fluorenone, then separate mixture by Column Chromatography (CC)

Pre-Lab Checklist

• Include balanced reaction equation:
  • $\text{Fluorene}\; (C<em>{13}H</em>{10}) + \tfrac{1}{2}\,O<em>2 \xrightarrow[\text{air}]{\text{NaOH / Aliquat 336}} \text{Fluorenone}\; (C</em>{13}H<em>{8}O) + H</em>2O$
• NEW required items (sections 6–8 per lab manual §3.2)
  • Chromatographic behaviour comparison (Rf’s vs. elution order) of starting material vs. product
  • Spectral features comparison (IR, UV, NMR) of fluorene vs. fluorenone
  • Detailed description of isolation & purification (“work-up”)
• PreLab Exercises
  1. Predict elution order from alumina column
  • Fluorene (non-polar, hydrocarbon) elutes FIRST
  • Fluorenone (polar ketone) retained longer (elutes second)
  • Justification: polar alumina (Al₂O₃) preferentially adsorbs polar compound via dipole–dipole/H-bonding; less polar species travels with non-polar solvent sooner
  1. Second low-Rf spot in fluorene TLC standard = fluorenone impurity formed by ambient oxidation (air/light) of fluorene

Column Chromatography (CC) Fundamentals

• Same partitioning principles as Thin-Layer Chromatography (TLC) but for preparative scale
• Flow direction difference
  • TLC: solvent ascends (capillary action) → more polar = lower $R_f$
  • CC: solvent descends (gravity/pressure) → more polar = longer retention, later elution
    ⇒ THINK “TLC picture is upside-down” when predicting orders

Stationary Phases

• Common: silica gel (SiO₂) & alumina (Al₂O₃)
  • Variants: different particle sizes, activities (I–III), acidic/basic modifications
• Choice guided by prior TLC, literature, or trial

Mobile Phase (Solvent) Selection

• Begin with very non-polar solvent; gradually increase polarity (“gradient”) to desorb analytes
  • Typical pairs: ligroin–CH₂Cl₂, hexane–ethyl acetate, hexane–toluene
• Avoid highly polar protic solvents (MeOH, H₂O) with normal silica/alumina — can dissolve the gel
• Mixing solvents macroscale produces heat; change polarity SLOWLY to prevent column cracking

Apparatus

• Range: pencil-thin microcolumns ➜ industrial (>1 m diameter)
• Microscale set-up (Fig. 8.1) components:
  • Glass column + fritted bottom plug + stopcock/valve
  • Clamp vertically; have multiple tared receiving vessels (1–3 mL fractions recommended)

Packing Methods

1. Dry-pack (microscale default)
   • Fill column with solvent, sprinkle dry adsorbent while tapping
   • Alternative dry-pack II: adsorbent in first, then solvent (for alumina only)
2. Slurry method (macroscale, best uniformity)
   • Pre-mix adsorbent with solvent to slurry; pour in carefully

• Critical: avoid cracks, air bubbles, channels; NEVER allow solvent front to drop below adsorbent (“running dry”)

Sample Loading & Elution

• Dissolve crude (minimum 5–10 drops polar solvent)
• Pipette onto flat adsorbent surface → aim for THIN horizontal band
• Add thin sand layer to protect surface, then continuously feed solvent
• Collect small fractions (≈$1{-}3\,\mathrm{mL}$) — easier to pool than to re-separate

Monitoring Progress

• Coloured compounds: watch visible bands
• Colourless compounds: use TLC — spot multiple fractions per plate; include standards
• Change solvent polarity to accelerate once non-polar band(s) recovered (e.g., switch from ligroin to 20 % then 50 % CH₂Cl₂ in ligroin)

Isolation & Post-Processing

• Pool identical fractions ↔ TLC/colour cue
• Evaporate solvent; on mg-scale usually no recrystallisation needed
• Flash chromatography
  • Apply ≈$10\,\text{psi}$ air/N₂ to speed flow; separation may worsen unless finer adsorbent used
• Reverse-phase (RP) chromatography
  • Silica surface derivatised with long alkyl (C-18) chains ⇒ stationary phase very non-polar
  • Mobile phase: polar (MeOH/H₂O, ACN/H₂O)
  • Elution order REVERSES: polar solutes elute first, non-polar retained
• Chiral chromatography
  • Stationary phase engineered with “handedness” → separates enantiomers (critical for pharma; e.g., Thalidomide case)

Experimental Procedure Details

Step 1 – Oxidation of Fluorene

• Reagents (10 mL Erlenmeyer, 1⁄2 inch stir bar)
  • $5\,\mathrm{mL}$ $10\,\mathrm{M}\;NaOH$ (⚠ corrosive)
  • $70\,\mathrm{mg}$ fluorene
  • $5\,\mathrm{mL}$ toluene
  • $\approx3$ drops Aliquat 336 (Stark’s catalyst, methyltricapryl ammonium Cl)
• Stir vigorously; monitor by TLC (20 % CH₂Cl₂/ligroin, UV 254 nm)
• When ~~50 % conversion (≈10 min) reached:
  1. Transfer to separatory funnel; rinse with extra $5\,\mathrm{mL}$ toluene
  2. Separate layers (note: toluene = organic upper?)
  3. Wash organic phase:
  • $5\,\%$ HCl $3\times5\,\mathrm{mL}$ (removes base & quaternary ammonium)
  • Sat. NaCl $3\times5\,\mathrm{mL}$ (brine)
  1. Dry over anhydrous $Na<em>2SO</em>4$; decant into 100 mL tared beaker
  2. Optional: gently reduce volume to $\approx1\,\mathrm{mL}$ on sand bath or just leave — toluene evaporates in hood
• Reaction rationale
  • Fluorene 9-position hydrogens unusually acidic (doubly benzylic)
  • $OH^-$ deprotonates → carbanion attacks $O<em>2$ forming hydroperoxide → loses $H</em>2O$ → ketone
  • Aliquat 336 transfers $OH^-$ into organic phase; avoids toxic Cr(VI) reagents

Step 2 – Column Chromatography of Mixture

• Column packed with $\approx4.5\,\text{g}$ Activity III alumina (height $\approx10\,\text{cm}$) in ligroin
• NEVER let solvent fall below adsorbent top
• Load crude via minimal 10 drops CH₂Cl₂ + 10 drops toluene solution
• Drain to surface; rinse/compact with ligroin until narrow band visible
• Add 4–5 mm sand; fill with ligroin
• Collect ≈10 fractions × $3\,\mathrm{mL}$ each in tared small vessels
• Analyze each fraction by TLC (20 % CH₂Cl₂/ligroin)
  • Spot 2–3 times → dry → UV
  • Four–five fractions / plate
• If yellow band (fluorenone) stagnates: switch to 20 % CH₂Cl₂/ligroin; later to 50 % mixture for final elution
• Combine like fractions, evaporate on sand bath (tilted), encourage crystallisation (scratch if oily)

Clean-Up & Waste Disposal

• Aqueous washes → sink with copious water
• Organic solvents → designated organic waste container
• When finished: drain column, remove bottom plug, leave wet column inverted in beaker; dry alumina disposed next period

Final Report & Evaluation

• Tape all developed TLC plates; mark spots
• Provide:
  • Mass & $\%$ recovery of unreacted fluorene
  • Mass & $\%$ yield of pure fluorenone (vs. reacted fluorene)
  • Calculated $R_f$ values, volumes, all weighing data
  • Discussion of TLC & CC quality (band sharpness, separation efficiency, solvent choices)
• PostLab Questions (prepare written answers)
  1. Effect of collecting larger-volume fractions?
     • Larger fractions risk co-elution → mixed compounds; poor resolution; may necessitate rerunning column.
  2. Why drop liquid level to alumina surface before sample application?
     • Ensures sample forms narrow band at interface; prevents dilution & premature movement → sharper separation.
• Grading rubric (total $100$ pts):
  • Observation/Data $20$
  • Calculations $12$
  • Results/Discussion $16$
  • Yields $24$
  • Chromatographic data interpretation $16$
  • PostLab questions $12$

Practical & Safety Notes

• NaOH is strongly caustic → wear gloves; wash immediately on contact
• Avoid splashing vigorously stirred biphasic reaction
• When mixing solvents inside column, heat may evolve; change polarity GRADUALLY
• Flash chromatography can accelerate but may lower resolution unless finer adsorbent used
• Fraction size rule: easier to combine small fractions than to separate large, mixed ones
• Never use MeOH or H₂O mobile phase on normal-phase silica/alumina — dissolves column