Liquid Chromatography
Chromatography: “Chroma” is Greek meaning “colour” and “graphein” is Greek meaning “to write”
Definition: To separate a mixture by relative attraction of its components as a mobile phase (a liquid or gas) passes over the stationary phase (a solid or liquid) as the sample travels through a packed column/tube.
Mobile phase: a liquid or gas which moves through the column/tubing
Stationary phase: is a solid or liquid which is part on the inside of the column or tubing the unknown solution is being passed through, this material is stationary/doesn’t move
Liquid Chromatography (LC):
1) Set-up laboratory equipment
2) Prepare the column (use a Pasteur pipette) plug one end with a small amount of cotton (use a wooden applicator stick to tamper down cotton)
3) Take a small, 10ml beaker and add silica. Pour the silica into the column by tipping the mouth of the beaker to the top of the column and using another pipette to direct the flow of the silica into the column
****Note: Different labs use different types of silica when packing column such as acid silica, silver nitrate silica, etc.
Other types of columns used: Carbon columns (packed in the same mannerism)
4) Pre-elute column: add your solvent to the column to saturate your column (mobile phase)
5) Monitor solvent levels: look for air bubbles, check for any leaks, etc.
6) Dry method (there is a wet method, but only recommended for expert users): pipette your entire sample directly on top of the packed column
7) After your sample has all started down the column, elute the column, forcing your sample through the column.
8) Once there is no more solvent on top of the column, elute the column again.
9) Analyze fractioned colours: compare with liked-colour fractions
10) Wash glassware and clean-up workstation
Definition: To separate a mixture by relative attraction of its components as a mobile phase (a liquid or gas) passes over the stationary phase (a solid or liquid) as the sample travels through a packed column/tube.
Mobile phase: a liquid or gas which moves through the column/tubing
Stationary phase: is a solid or liquid which is part on the inside of the column or tubing the unknown solution is being passed through, this material is stationary/doesn’t move
Liquid Chromatography (LC):
1) Set-up laboratory equipment
2) Prepare the column (use a Pasteur pipette) plug one end with a small amount of cotton (use a wooden applicator stick to tamper down cotton)
3) Take a small, 10ml beaker and add silica. Pour the silica into the column by tipping the mouth of the beaker to the top of the column and using another pipette to direct the flow of the silica into the column
****Note: Different labs use different types of silica when packing column such as acid silica, silver nitrate silica, etc.
Other types of columns used: Carbon columns (packed in the same mannerism)
4) Pre-elute column: add your solvent to the column to saturate your column (mobile phase)
5) Monitor solvent levels: look for air bubbles, check for any leaks, etc.
6) Dry method (there is a wet method, but only recommended for expert users): pipette your entire sample directly on top of the packed column
7) After your sample has all started down the column, elute the column, forcing your sample through the column.
8) Once there is no more solvent on top of the column, elute the column again.
9) Analyze fractioned colours: compare with liked-colour fractions
10) Wash glassware and clean-up workstation