Lab J: Isolation of Bixin from Annatto Seeds using Column Chromatography (Part II)

What is chromatography?

• Separation of compounds based on polarity.

• Most common organic purification technique.

What is column chromatography?

• Separates macroscopic amounts of compounds

• Like running a “larger TLC”

How do you choose a suitable solvent?

• Use TLC to determine appropriate solvent for separation.

• You want a solvent such that desired compound will move off the baseline + separate well from other compounds

• Choice of solvents:

  – Dichloromethane (DCM)

  – 3 % Ethanol in Dichloromethane (3% EtOH in DCM)

  – 10 % Ethanol in Dichloromethane (10% EtOH in DCM)

Packing the column reminders?

• Make sure the Column is clamped in the middle and it is vertical.

• Prepare (in a beaker) a slurry of silica gel and the appropriate solvent.

• Add slurry via POWDER FUNNEL.

• Add more solvent to wash slurry down the sides of the column using a pipette.

• Open stopcock to collect the solvent in a beaker and reuse solvent (as long as it’s not colored).

• DO NOT let solvent level fall below the top of the silica.

Reminders when preparing the sample?

• Sample needs to be COMPLETELY DISSOLVED. Use pipette to dissolve as much sample as possible.

• Load sample in a narrow band onto the column.

How do you avoid broad bands?

• Use as concentrated a solution of the extract as possible but load all your product onto the column.

• Make sure the extract is fully loaded into the column before adding solvent all the way to the top.

• This process usually takes a few rounds of adding the 3-4 mL of solvent in order to push the colored bands down the column.

Why should you not let the column run dry?

• Make sure there is always solvent above silica gel.

• If dry, cracks develop inside silica gel in column and affects separation.

What will the fractions look like?

• Yellow fractions will come off the column first. Collect these in a 125 mL Erlenmeyer flask.

• The intense Orange-red (Bixin) band will be next.

• When the Bixin band has finished eluting – you should stop collecting fractions

Waste disposal?

• After combining fractions to be rotovaped - drain columnwaste into Erlenmeyer flask

• After draining, turn column upside and place 4 weigh boats below.

• Test tubes w/ unused fractions —> C,H,O halogenated waste container

Safety hazards?

Silica gel is an inhalation hazard.

Dichloromethane is a carcinogenic compound.