School of Chemistry Safety Protocol – Preparative Column Chromatography
Protocol for the Use of Cryogens
Protocol for the Use of Cryogens
PROTOCOL FOR PREPARATIVE COLUMN CHROMATOGRAPHY
Main Hazards
•Chemical Hazards Associated with the Solvents and Reaction mixtures
•Chemical Hazards Associated with the Stationary Phase (usually silica)
•Rupture of Glass columns on over pressurisation.
Key Safety Procedures
Ensure COSHH and Risk assessment on the solvents, reaction mixtures and stationary phase have been performed.
Wear Safety Glasses
Work in a Fume Hood
Do not over pressurise column
Wear gloves
General Comments
Ensure COSHH and Risk assessment on the solvents, reaction mixtures and stationary phase have been performed. These assessments will normally require that suitable gloves are worn whilst performing column chromatography and that it is performed in a fume hood. Advice on glove types is available from the Kimberley Clarke website. Stationary phases such as silica should only be handled in a fume hood and be disposed of in special waste containers. Wear Safety Glasses. Do not over pressurise columns. Perform chromatography in line with the following protocol.
Detailed Instructions for Use
Carefully check the column for the presence of cracks, especially small star cracks.
Clamp column vertically. Avoid the use of clamps with bare metal jaw as these can fracture the glass. Do not over tighten the clamps. For large columns, support the weight of the column by using a separating funnel ring clamped under the column.
Place a small piece of cotton wool in the column just above the tap using a glass rod. This is not required for columns with an integral sinter.
Pack the column in accordance with method A or B.
Method A, Slurry packing: General for all types of chromatography.
Measure dry stationary phase into the column. Silica chromatography is normally performed on a column approx. 15 cm in length.
Pour stationary phase into a conical flask which is at least 5x the volume of the stationary phase.
In a fume cupboard. Away from ALL IGNITION SOURCES, pour solvent mixture onto the silica and stir until all air is released and a fluid slurry is formed.
(This step is not necessary if the column has an integral glass sinter). Place a layer of sand in the base of the column to cover the cotton wool. Ensure that this layer of sand reaches above the point where the column starts narrowing towards the tap. Close the tap and place 4 cm of solvent in the column to protect the sand.
Agitate the slurry and pour into the column in a smooth action, Use a funnel if necessary. Rinse the remaining slurry from the flask and pour into the column. Allow the slurry to settle in the column. Connect a pressurisation device to the top of the column (e.g. approved column pump or hand bellows). Clip the glass connector in place with a plastic clip, place an empty receptacle under the tap, open the tap and gently pressurise the column. Do not over pressurise the column as it will fracture (if in doubt use a column manometer), however with sensible use of hand bellows or column pump on undamaged columns it is very hard to actually break the column in this way. Pump the solvent through the column until the level just touches the top of the silica.
Method B, Dry Pack. Do not use this method for stationary phases which expand significantly on solvent addition (e.g. ion exchange resins).It is also not recommended for silica and ether combinations as the solvation of the silica with ether is very exothermic.
(This step is not necessary if the column has an integral glass sinter.) Place a layer of sand in the base of the column to cover the cotton wool. Ensure that this layer of sand reaches above the point where the column starts narrowing towards the tap.
Measure dry stationary phase into the column. Silica chromatography is normally performed on a column approx. 15 cm in length.
Add the solvent mixture to the column. Connect a pressurisation device to the top of the column (e.g. approved column pump or hand bellows). Clip the glass connector in place with a plastic clip, place an empty receptacle under the tap, open the tap and gently pressurise the column. Do not over pressurise the column as it will fracture if in doubt use a manometer, however with sensible use of hand bellows or column pump on undamaged columns it is very hard to actually break the column in this way. Pump the solvent through the column until all air is expelled and the silica has a homogeneous appearance (This may require several column volumes). Continue to pump solvent through the column until the level just touches the top of the silica.
Loading the Column
Standard Method: Dissolve the compound mixture in the minimum volume of solvent, ideally the same solvent mixture as the eluent. Other solvents can sometimes be used. Gently pipette the mixture onto the surface of the silica, taking care not to disturb the column. Using fresh solvent, rinse the flask and sides of the column. Pump the solvent through the column until the level just touches the top of the silica. Gently add about 1 cm of sand to the top of the column and then add about 4 cm of solvent to the column and pump through. The column can now be filled with solvent and the chromatography started.
Some researchers prefer to add the sand before the compound and this method is perfectly acceptable.
Alternative Method: Pre-absorption of compounds. If the mixture is not soluble in the eluent it is sometimes useful to pre-absorb the mixture on stationary phase. Dissolve the mixture in any volatile solvent and add dry silica to the mixture, enough to form a layer of about 1 cm on the column. Evaporate the solvent on a rotary evaporator using a sintered adaptor to prevent the dry silica entering the apparatus. Once the silica is dry and freely flowing it can be placed directly on top of the column and then moistened with a little solvent. Sand is then added and chromatography continued as normal. If on evaporation the silica is not freely flowing, add more solvent and silica and repeat the process.
Running the Column
Collect fractions in test tubes held below the column. Do not allow the level of solvent to drop below the level of the sand as this will introduce air into the column. Always release the pressure before closing the tap.
After the column is completed.
Pump all remaining solvent out of the column. Dispose of waste solvent in the appropriate waste container. To remove remains of the solvent either leave attached to the column pump until the silica is dry or suck the column dry using a water pump (Connect the column outlet to the water pump with a piece of rubber tube and via a trap to prevent the solvent entering the drain, apply a gentle vacuum until the silica is dry and can be tipped out of the column).
Dispose of the silica in the waste silica bins unless it is contaminated with dangerous or smelly compounds in which case it should be stored separately. Contaminated silica should be treated to provide waste in its least harmful form.
Emergency Procedures
In case of contact with solvents or silica wash exposed areas thoroughly with water. Seek medical advice if the COSHH assessment on the material concerned requires such treatment.
Protocol written by: / S. L. WarrinerApproved by the Safety Committee: / 5. 2001
Last Review by: / R. Fox
Last Review Date: / 7. 2015
The School of Chemistry Safety Committee have approved this protocol for the named School operation or procedure. This task must be carried out in line with this protocol. If you believe that this protocol is unsuitable for the circumstances in your laboratory or area of work discuss this procedurewith your supervisor or line manager and the appropriate Safety Advisor. Any modified operating procedures must be documented, approved and signed by your supervisoror line manager before they are undertaken
Page 1 of 4