Column Chromatography

A reaction in an organic chemistry lab rarely produces a pure product. There’s usually unreacted starting material, by-products and reagents mixed in. Column chromatography is one of the main ways chemists separate that mixture and collect the product in pure form. Where thin-layer chromatography tells you what’s in a mixture, column chromatography lets you actually take the components apart and keep them.

It’s the same technique the botanist Mikhail Tsvet used in 1903 to separate plant pigments, and it’s still used every day in research and industry.

The principle

A glass column is packed with a stationary phase, usually silica gel (or sometimes alumina). The mixture is loaded at the top, and a solvent (the eluent, or mobile phase) is passed down through the column.

As the eluent flows, components move down at different speeds:

• On silica, non-polar compounds are held weakly and move quickly.
• Polar compounds stick more strongly to the silica’s Si–OH groups and move slowly.

The mixture separates into bands. The eluent coming out of the bottom (the eluate) is collected in a series of test tubes or flasks called fractions. Each band ends up in different fractions, which are then analysed and combined.

For the general theory, see chromatography.

Step 1: choose conditions with TLC

Column chromatography is planned with thin-layer chromatography, because TLC uses the same stationary phase and gives a fast preview.

1. Run TLC of the crude mixture in several eluent mixtures (for example, different ratios of hexane and ethyl ethanoate).
2. Choose an eluent that gives the desired compound an Rf of about 0.25–0.35, well separated from other spots.

A lower Rf on TLC means the compound will take longer to come off the column; a very high Rf means it will come off too quickly to separate.

Step 2: prepare the column

Equipment: glass column with a tap, silica gel, sand, cotton or glass wool, eluent, collection tubes, clamp and stand, funnel. A fume cupboard is essential because large volumes of organic solvent are used.

1. Clamp the column vertically. A tilted column gives slanted, overlapping bands.
2. Plug the bottom with a small piece of cotton or glass wool (unless the column has a built-in frit), then add a thin layer of sand to give a flat base.
3. Pack the silica. Two common methods:
   • Slurry (wet) packing: mix the silica with eluent to make a slurry and pour it in, tapping the column gently to settle it evenly and remove air bubbles.
   • Dry packing: pour dry silica in, then run eluent through. A typical ratio is about 30–100 g of silica for every 1 g of crude mixture, depending on how difficult the separation is.
4. Add a layer of sand on top of the silica to protect the surface when adding solvent.
5. Never let the solvent level drop below the top of the silica. If the column runs dry, cracks and channels form, and separation is ruined.

Step 3: load the sample

The mixture must enter the column as a narrow band at the top. A wide starting band gives wide, overlapping bands later.

• Wet loading: dissolve the sample in the smallest possible volume of eluent (or a slightly less polar solvent) and add it carefully to the top with a pipette. Open the tap to let it soak into the silica.
• Dry loading: dissolve the sample, mix it with a little silica, evaporate the solvent to leave a free-flowing powder, and pour that onto the column. This is useful for samples that don’t dissolve well in the eluent.

Step 4: elute and collect fractions

1. Carefully fill the column with eluent without disturbing the top layer.
2. Open the tap and start collecting fractions of equal volume in numbered test tubes.
3. Keep topping up the eluent so the column never runs dry.
4. If compounds are coloured, you can watch the bands move and separate. Most are colourless, so fractions must be analysed.

Gradient elution: sometimes the eluent is made gradually more polar during the run (for example, from 10% to 30% ethyl ethanoate in hexane). This moves strongly retained polar compounds off the column faster once the less polar ones have been collected.

Step 5: analyse the fractions

Spot every few fractions on a TLC plate, alongside the crude mixture, and visualise:

• Fractions containing only the spot of the desired compound are pure and are combined.
• Fractions containing two overlapping compounds are kept separate (and may be re-purified).
• Fractions with nothing are discarded.

Step 6: recover the product

Combine the pure fractions and remove the solvent, usually with a rotary evaporator, which evaporates solvent quickly under reduced pressure at a low temperature. Weigh the product to calculate the yield, and check its purity (for example, by melting point, TLC or spectroscopy).

Flash chromatography

In traditional gravity columns, solvent drips through slowly. In flash chromatography, introduced in 1978 by W. Clark Still and colleagues, gentle air or nitrogen pressure is applied to the top of the column to push the eluent through much faster, with finer silica. It’s now the standard method in most organic labs, and automated flash systems with pre-packed cartridges and UV detectors are common.

Common problems

Problem	Cause	Fix
Bands overlap	eluent too polar; column overloaded; sample band too wide	use a less polar eluent, more silica, a narrower sample band
Compound never comes off	eluent not polar enough	increase eluent polarity (gradient)
Bands are crooked	column not vertical or unevenly packed	clamp vertically; pack carefully
Cracks in the silica	column ran dry, or heat from solvent mixing	never let it run dry; pre-mix eluents
Everything comes off together	eluent far too polar	choose a weaker eluent based on TLC
Product decomposes	acid-sensitive compound on acidic silica	use neutral alumina or add a little base (e.g. triethylamine) to the eluent

Other kinds of column

• Alumina columns: alternative polar stationary phase, available as acidic, neutral or basic.
• Reversed-phase columns: non-polar bonded silica (as in HPLC) for polar compounds.
• Ion-exchange columns: separate charged molecules such as amino acids and proteins.
• Size-exclusion (gel filtration) columns: separate molecules by size, widely used for proteins.

Safety

• Work in a fume cupboard; organic solvents are flammable and their vapours are harmful.
• Silica dust is a respiratory irritant: weigh and handle it carefully, ideally in a fume cupboard, and consider a dust mask if required by your risk assessment.
• Take care if using pressure: use equipment designed for flash chromatography and never pressurise ordinary glassware beyond its rating.
• Dispose of solvent waste and used silica as directed.

Key takeaways

• Column chromatography purifies compounds by passing an eluent through silica (or another stationary phase) and collecting fractions.
• On silica, non-polar compounds elute first; polar compounds are held longer.
• Choose the eluent using TLC so the desired compound has an Rf of about 0.25–0.35.
• Pack the column evenly, load a narrow sample band, and never let the column run dry.
• Analyse fractions by TLC, combine the pure ones, and remove solvent to recover the product.