Column chromatography is the most widely used purification method in organic chemistry. It separates compounds based on their differential partitioning between a mobile phase (solvent) and a stationary phase (adsorbent).

Stationary Phases

• Silica gel (SiO₂): the most common stationary phase. It is polar and retains polar compounds more strongly. Particle sizes range from 40–63 µm for flash chromatography to 15–40 µm for HPLC.
• Alumina (Al₂O₃): available in basic, neutral, and acidic forms. Less common than silica but useful for base-sensitive compounds and separations where silica causes degradation.
• C18 reverse-phase silica: silica modified with octadecyl (C18) chains, making it non-polar. Used with polar mobile phases (water, methanol, acetonitrile). Reverse-phase chromatography is the standard for separating non-polar to moderately polar compounds.

Mobile Phase (Eluent)

The solvent system is chosen by thin-layer chromatography (TLC). The goal is an Rf of 0.2–0.4 for the target compound. Start with a non-polar solvent (hexane, petroleum ether) and add a polar modifier (ethyl acetate, methanol) in increasing proportions. Gradient elution — starting with low polarity and increasing gradually — gives the best separation.

Flash Chromatography

Flash chromatography uses compressed air or nitrogen to push solvent through the column at 1–10 bar, reducing separation time from hours to minutes. Modern flash systems (Biotage, Teledyne Isco) use pre-packed cartridges with a gradient pump and an integrated UV detector that automatically collects fractions when a peak is detected.

Manual flash setup:

1. Pack a glass column with silica — either dry-packed (tap to settle) or slurry-packed (pour a silica–solvent suspension).
2. Apply the sample as a concentrated solution or adsorbed onto a small amount of silica (dry load).
3. Add a layer of sand on top to protect the silica surface.
4. Apply solvent pressure and collect fractions in tubes.
5. Analyze fractions by TLC. Combine pure fractions and evaporate.

Fraction Collection and Detection

Fractions are typically 5–20 mL depending on column size and separation difficulty. TLC is the standard method for determining which fractions contain the desired compound. UV-active compounds can be detected under a 254 nm UV lamp. Staining the TLC plate with KMnO₄, p-anisaldehyde, or ceric ammonium molybdate visualizes non-UV-active compounds.

Normal vs. Reverse Phase

Normal-phase (silica, non-polar mobile phase) separates by polarity — non-polar compounds elute first. Reverse-phase (C18, polar mobile phase) separates by hydrophobicity — polar compounds elute first. Reverse-phase is dominant in analytical HPLC but less common in preparative organic purification, where normal-phase silica remains the standard.