OHS026
Safe Work Procedure
Faculty/Division
Medicine
School/ Divisional Unit
School of Medical Sciences/IIRC
Document number
24_SWP_HPLC
Initial Issue date
12-March-2010
Current version
1.0
Current Version
Issue date
12-March-2010
Next review date
12-March-2011
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this
form.
Safe Work Procedure Title and basic description
Title: Operation
of high performance liquid chromatography machines
Description: This SWP applies to the high performance liquid chromatography (HPLC) machines in Lab 524
in the Wallace Wurth Building
Associated risk assessment title and location: Medicine server: Files-1-med/IRU/Common/Risk assessments
Describe the activity or process
Please do not use these machines unless you have been trained in their use. For each HPLC, follow
additional safety information in the manual.
1. Procedure
- Open the tap on the helium bottle to release helium into the lines
- Switch on the two components of the HPLC, the pump and the absorbance detector (or fluorescence
detector if connected)
- Check that there is sufficient solvents in the reservoirs on top of the HPLC to complete your intended
run and ensure that the sampling and sparging tubes lines well submerged in the solvent
- If you need to refill the reservoirs do so by taking the bottle down and pouring the liquid in on the
bench (or in the fume hood for acetonitrile), while doing this avoid laying the sampling and sparging
lines on the HPLC or bench where they could pick up dust, instead lay them on a clean weigh boat or
other clean surface. After filling return the sampling and sparging lines to the bottles and ensure that
you cover the solvent reservoirs to prevent dust accumulating in them, then replace on the top of the
HPLC
- Switch on the computer and load the Millenium program, then load your method set
- Ensure that the eluate line is flowing into a large volume waste bottle (eg 2 litre side-arm flask)
- Always filter samples before loading since protein aggregates, agarose beads or other particulate
matter will clog the columns and make them unusable
- Inject samples by switching the injection port to “load”, then insert the Hamilton syringe, inject the
sample and switch the injection port to “inject”. Inject samples using only the blunt ended Hamilton
syringes near the HPLC, do not use other needles as this could damage the machine
2. Clean up procedures
- After completion of the experiment, empty the contents of the waste bottle into an appropriate
bunded HPLC waste container.
- Make sure the waste container is labeled with the full chemical names of all components, the
concentration of the components and information regarding packaging group, and dangerous goods
class. Note: Full containers must not be stored in the main lab (see Waste disposal procedure below).
- Switch off the machines and the computer
- Turn off the helium at the tap on the bottle
3.
-
Waste disposal procedure
Full HPLC waste containers should be disposed of as chemical waste.
Place the container in the tubs in room 525 (histology area).
Log the bottle on the chemical waste inventory form and attach a filled-in disposal label (both of
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Safe Work Procedure
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Date Effective: 01/01/2007
Current Version: 1.2, 15/08/2007
-
these are provided in room 525).
Make sure that:
The chemical waste container is not leaking and there is no spillage on the exterior of the
container.
The container is labelled without abbreviations
Waste is segregated, if necessary, in accordance with Dangerous Goods Class.
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
HPLC system (pump and detectors)
Solvents (most commonly acetonitrile and trifluoroacetic acid but it will depend on the specific protocol)
Lab coat
Gloves - Use the blue nitrile gloves, as these have higher solvent resistance
Enclosed shoes
List potential hazards and risk controls including
specific precautions required
Acetonitrile is highly flammable, do not use in vicinity of ignition sources
Acetonitrile is toxic by inhalation and is absorbed through skin, use the fume hood while handling and
always wear gloves and a lab coat
Trifluoroacetic acid is highly corrosive, use the fume hood while handling the concentrated stock
Review the MSDS for both acetonitrile and trifluoroacetic acid before using
List emergency shutdown instructions
In case of spill use absorbant to clean up, follow the instructions in the MSDS (Spill kit is located near the
entrance to the laboratory on the left hand side)
In case of spill onto electrical components switch off immediately at the power point. Inform the plant
custodian.
List clean up and waste disposal requirements
See above
In general, keep the area around the HPLC tidy
List legislation, standards and codes of practice used
in the development of the SWP
Acetonitrile MSDS
Trifluoroactetic acid MSDS
AS/NZS 2243.2:2006 Australian/New Zealand Standard™ Safety in laboratories Part 2: Chemical aspects
Supervisory approval, training, and review
Supervisor:
Signature:
Plant custodian: Jesse Goyette
Signature
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Date Effective: 01/01/2007
Current Version: 1.2, 15/08/2007
List competency required:
All users must have completed the Laboratory safety course and be instructed in the use of the centrifuge prior to use.



UNSW Laboratory Safety Awareness training
UNSW Occupational Health & Safety Awareness training
UNSW Hazardous Substances training

Demonstration of the techniques by a qualified individual is required
SWP review date: 12-March-2011
Responsibility for SWP review: Plant custodian
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Safe Work Procedure
Uncontrolled document when printed
Date Effective: 01/01/2007
Current Version: 1.2, 15/08/2007