OHS026
1. Completed by: Dr. Pall Thordarson and
A/Prof. Jonathan Morris
Safe work procedure
Staff/Student number:
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this form
Faculty/Division:
Faculty of Science
Document number
F10-SWP-304
School/Unit:
School of Chemistry
Initial Issue date:
16-August 2011
Current version:
V1.1
Current Version
Issue date: 19-August 2011
Next review date: 19-August 2013
2.Safe work procedure title and basic description of activity
Title: Flash Column Chromatography using glass columns (under pressure)
Description of activity: Proper usage of flash column chromatography (silica or alumina)
3.List Hazards and risk controls as per risk assessment
Associated risk
assessment
number and
location:
F10-RA-304
See School of
Chemistry
Safety Page and
Faculty of
Science
Database
Hazards:
Silica or alumina dust
Controls
Proper training, consult MSDS for silica or alumina. The silica used for flash
chromatography is an extremely fine dust that will damage the lungs.
Alumina dust is also toxic and harmful to the respiratory system.
Appropriate training. All handling of dry silica or alumina must be carried
out in a fume hood. Avoid breathing dust. Use appropriate PPE including
protective safety glasses and a laboratory coat and especially chemically
compatible gloves at all times Use facemask (type: AS 1716 P1) for larger
(> 50 g) quantities.
Implosion or explosion
of glassware under
pressure
Appropriate training. Inspect glassware prior to use for cracks/faults. Make
sure that column is securely clamped. Only use in ventilated fume
cupboard. Always pull fume cupboard sash down when this is possible.
Correct use of pressured gas – use only very gentle pressure / flow. Use
gloves and other appropriate PPE.
Other physical injury
due to glassware
Appropriate training. Inspect columns, sample and collection vials for
cracks before use and replace if faulty. Show extra care with adapters used
to apply pressure on column and when fitting these with tubes for nitrogen
gas. Correct use of glassware. Use gloves, preferably leather and other
appropriate PPE such as protective safety glasses and a laboratory coat.
Spillage and
contamination from
chemicals used to run
the flash column, Fire
and explosion
Consult MSDS for all chemicals used and fill in the corresponding School of
Chemistry Risk assessment form. Appropriate training. First aid on site.
Wear proper PPE, especially chemically compatible gloves and protective
eyewear. Dilute corrosive substances if possible. Avoid ignition sources.
Carry all work out in a fume hood. Clean up spillages immediately.
Asphyxiation from
nitrogen gas.
Appropriate training. Perform flash chromatography in a fume hood. Ensure
to close nitrogen source off immediately after use.
4.List resources required including personal protective clothing, chemicals and equipment needed
Materials and Equipment
• Glass column for chromatography adapter (solvent reservoir optional).
• Flash chromatography grade silica or alumina. Acid-washed sand (optional)
• Solvents to run column with (hexane, ethyl acetate, dichloromethane and methanol the most common ones)
• The sample to run. Pipettes if required for transferring sample.
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• Tube(s) for nitrogen flow, funnel(s), cotton to plug the column if necessary, aluminum foil (to cover column if material is
light sensitive)
• Collection vials for the eluent / products from column.
Personal Protective Clothing and Equipment
• Protective safety glasses
• Laboratory coat
• Gloves (choose according to chemical compatibility and/or physical hazard)
• Facemask (if required), minimum AS 1716 P1 type.
5.List step by step instructions or order for undertaking the task
1. Preparing and packing the column
•
This procedure MUST be done in a fume hood! Use of a face mask is recommended for larger quantities (> 50
g).
•
•
Only suitably trained persons can do flash column chromatography.
Choose the appropriate column size for your separation. Consult your supervisor if unsure. W. Clark Still, M. Kahn and
A. Mitra (J. Org. Chem., 1978, 43, 2923) also provide some advice on this. Longer columns are not necessary better –
this needs to be balanced with how well you can control the flow rate to be at optimum (Van Deemter plot – see e.g.,
http://www.reachdevices.com/SetUpColumn.html) with 20 cm more than sufficient in many cases. Focus rather on the
appropriate diameter for your column to give the optimum sample-to-silica (or alumina) ratio.
Some columns come with a pre-fabricated frit at the bottom. These make loading easier but not favoured by all as this
may increase the dead volume. If your column is not fitted with a frit, you need to plug the bottom. Make a cotton plug
and insert at the bottom of the column with the tap closed. To push the plug in you may have to use a designated
metal or glass rod – take extra care not to break the column doing this and if using a glass rod, don’t apply too much
pressure or touch the end of the rod. Don’t use excessive force to push the plug in. Good hand protection (e.g., leather
gloves) is strongly recommended.
Clamp the column in position. If desired, you may want to add a layer of sand on top the cotton plug or frit. Add a little
bit of your first eluting solvent if desired to wet the plug / sand.
Prepare the gas inlet adapter. The exact way you do this depends on the set-up in your laboratory. Ensure that there
is some way of controlling the pressure and allow excess pressure out to eliminate the risk of explosion once the
column system is fully assembled. The pressure is usually controlled in two stages: i) at the gas source (in-house or
cylinder) and ii) somewhere close to the column. Turn the gas flow at the source on to the lowest level possible –
NEVER turn the gas valve at the source to the maximum level possible. For the second stage a precise pressure valve
is preferable. If not available, one solution is to use a three-valve T-junction on the gas hose with one free; the gas
pressure can then be adjusted and quickly released by putting a finger on the open end of the T-junction.
To pack the column there are essentially two different procedures; i) slurry packing and ii) dry packing. The choice
between these will depend on the circumstances of your separation problem and personal preference. Consult
supervisor if unsure.
i)
Dry packing: Measure out the required amount of silica or alumina from its container in a fume hood into a
beaker or similar vial. Add solvent to the column to a level little above the desired length of your packed
column. Using a dry funnel, sprinkle the silica or alumina into the solvent. Tap the column gently with a pencil
to help the silica or alumina to pack and allow solvent to drain from the column into a conical flask to prevent
overflowing.* Let the silica or alumina settle and drain the solvent using GENTLE pressure of nitrogen until the
solvent level is just even with the surface of the silica or alumina.**
ii)
Slurry packing: Measure out the required amount of silica or alumina from its container in a fume hood into a
conical flask or a similar vial. Add your solvent (typically 2-3 mL per gram for alumina but up to 10 mL per
gram for silica) of the silica or alumina and swirl around gently until a uniform slurry is obtained with no visible
dry pockets or air-bubbles. Add more solvent if necessary. Note when using alumina that this procedure can
be quite exothermic. Fill your column with solvent to a level above the desired packed column length. Using a
funnel, quickly pour the slurry into the column. Place an conical flask under the column, open the tap and
allow the liquid to drain into it but don’t let it drain below the level of your silica or alumina.* Continue to
transfer the slurry to the column until all the silica or alumina is added. Add more solvent if necessary. When
finished packing, drain the excess solvent using GENTLE pressure of nitrogen until the solvent level just
reaches the top level of the of the silica or alumina.*/** Close the tap. Your column is now "packed”.
•
•
•
•
*In many circumstances you can reuse this solvent – consult your supervisor for advice is this the appropriate
course of action for your system.
**If desired, you may want to add a layer of sand on top of the silica to the surface sturdier (n.b., this may
though decrease your separation power). If you are planning to do this, don’t drain the solvent completely,
leave ca 3-6 cm layer of solvent, sprinkle the dry sand gently via funnel into the column. Once the sand has
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settled, drain the excess solvent using GENTLE pressure until the solvent level just reaches the top level of
the silica (it is OK if the sand is partly dry).
•
•
If you are working with light-sensitive materials, cover the outside of the glass column with aluminum foil up to the top
level of the silica or alumina.
Unless you stay with the column at all times, you also need to identify the content of the column (solvent, sample,
silica or alumina) by attaching a label directly on or near the column.
•
2. Loading the sample
The following procedure MUST be carried out in a fume hood!
•
•
Only suitably trained persons can do flash column chromatography.
To load the sample there are essentially two different procedures; i) wet loading and ii) dry loading. The choice
between these will depend on the circumstances of your separation problem and personal preference. Wet loading is
ideal when your sample is freely soluble in the solvent you use to start the column with while dry loading is preferable
if the sample is not soluble in the solvent you start with. Consult supervisor if unsure.
i)
Wet loading: Test if you sample is soluble in the solvent you use to pack the column with and you indent to
start the column with. N.b., using a different solvent to dissolve your sample than used to pack/elute the
column with can severely affect your separation. Dissolve the sample you have with minimum amount of
solvent (generally < 5 mL). Carefully use a Pasteur pipette or a syringe with a wide needle to transfer the
solution to the column. Addition must be carried out in such a manner that the silica surface is not disturbed
(even when a protective layer of sand is used). Use 1-2 mL of solvent to rinse your sample vial and transfer
this solution also to the column in the same manner.
ii)
Dry packing: In a suitable large round-bottom flask, dissolve your sample in a suitable solvent (this does not
have to be the same solvent as you use for eluting the column) and add to this solution about 1/20 to 1/10 of
amount of dry silica (dry packing is rarely used with alumina columns) you used to pack the column. Add more
solvent if necessary until you form a uniform slurry. This slurry is then gently evaporated on a rotary
evaporator which leaves a dry batch of silica saturated with the sample to be separated. It should be a free
flowing powder. If it is still an oil, add more silica and repeat the procedure. After this mix is prepared,
additional eluant is poured into the silica column to get about one inch of eluant above the silica. The powder
is then added, making sure to always retain a small (1.5-3 mm) eluant layer (if all the eluant getting absorbed
by this addition more eluant must be added). The goal is to form bubble-free slurry over column packing
without much disturbance. This does require practice.
•
Whether you use the wet or dry loading method, the last step is to lower the solvent again down to just above (1.5-3
mm) the level of silica or alumina using GENTLE pressure. Fresh eluant is added then so that its level reaches about
10 mm and this is drained again to just above the silica or alumina level (1.5-3 mm). This step is repeated one or two
more times. The column is now ready for eluting which should be done immediately to avoid accidental solvent
draining below the top silica layer due to leaks or evaporation of the solvent.
•
•
•
•
•
•
3. Running the column and finishing up
This procedure MUST be carried out in a fume hood.
Only suitably trained persons can do flash column chromatography.
Add as much as you desire of your eluant gently (or as much as fits). Start eluting the column. The flow rate is
adjusted by the column's stopcock and gas pressure. If you are using a pressure gauge, set the pressures between 14 psi depending on silica size (lower for large particle size) and solvent (higher for more viscous solvent). For glass
column 7 psi is the ABSOLUTE HIGHEST limit.
Collect your fractions in the appropriate vials. Keep these if possible in the fume hood while determining which fraction
you wish to isolate/combine and then transfer to round bottom flask(s) for removing the solvent (if desired). If you need
to keep the vials for prolonged periods, cover with aluminum foil and leave in a fume hood.
When you have completed your run it is good practice to drain all the solvent of the column using gentle pressure.
To empty the silica or alumina from the column the use of a facemask 1716 P1 type is highly recommended and
ALWAYS REQUIRED FOR LARGER QUANTITES (> 50 g). Start this procedure by placing the column upside down
above a large beaker (several times the volume of the silica used) AS FAR AS POSSIBLE AT THE BACK OF A FUME
HOOD with the sash down to minimize exposure to the silica or alumina dust. If the silica or alumina is already dry it
may fall out quickly under gravity. Otherwise leave it either to dry and fall out over a period of time (e.g., overnight) or
put a nitrogen hose on the stopcock end of the column and apply VERY GENTLE pressure to push the silica or
alumina down out of the column.
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6.List emergency shutdown procedures
Mop up spilt silica or alumina with damp cloth. If silica or alumina is inhaled move to fresh air and obtain medical
assistance. In case of solvent spillage, do not operate switches nearby and mop up solvent if in fume cupboard. If spillage
occurs outside fume hood, evacuate area and allow to evaporate. Turn off nearby power at power point. If unable or
unsafe to do so use the emergency cutoff switch (red button) for the laboratory (e.g., lF10-234) usually located on the wall
near the entrance to the lab.
7.List Emergency procedures for how to deal with fires, spills or exposure to hazardous substances
Contain the fire or spillage if safety of all personnel is not compromised, otherwise shutdown the power using the
emergency cutoff switch (red button) for the laboratory (e.g.,, lF10-234) and call the security for emergency (x56666).
8.List Clean up and waste disposal requirements
Disposal of used silica or alumina should be into an appropriately labelled solid-waste container.
•
Consult MSDS and place any solvents or chemicals used in the appropriate waste containers; non-halogenated
solvents/chemicals in non-halogenated waste and halogenated solvents/chemicals in halogenated waste
9.List legislation used in the development of this SWP
NSW OHS Act 2000, NSW OHS Regulation 2001, Code of Practice for the Labelling of Workplace Substances
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
10a.List competency required – qualifications, certificates, licensing, training - e.g. course or instruction:
All personnel operating this equipment must have training and instructions on the safe use of the equipment and they
must be assessed as competent.
10b. List competency of Assessor
Academic supervisor, post-doctoral fellow or senior PhD student (minimum 1 year into PhD).
11.Supervisory approval, And review
Note: Local supervisor must determine appropriate authorisation and final sign off when this document is
downloaded.
Supervisor:
Signature:
Responsibility for SWP review:
Date of review:
12.SWP Sign off sheet
SWP name and version:
In signing this section the assessor/ authoriser agrees that the following persons are competent in following this
SWP
Name
Signature
Date
Competent
Name of Assessor/Authoriser
Assessor/Authoriser
signature
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