Heparin
EZ-Link NHS-LC-Biotin
Hazardous Substances Policy - Assessment
CHEMICAL HAZARD AND RISK ASSESSMENT
School of Biosciences
Name of supervisor / Dr.J.E.Turnbull / Assessment Number* / JETLW16#2070
Signature
Assessor / Laine Wallace / Date of Assessment / 20/08/02
Signature
NotesA School COSHH form in Word is available on the School Server.
Available from the Health and Safety Unit.
Guidance on making an assessment is given in Making a Chemical Hazard and Risk Assessment.
Guidance is also available from Guidance on Completing the Chemical Hazard and Risk Assessment Form.
Use a continuation sheet to expand any section of this form in hard copy version.
1 / LOCATION OF THE WORK ACTIVITY / 5th Floor Biosciences Tower2 / PERSONS WHO MAY BE AT RISK
List names where possible / Laine Wallace, Dr.J.E.Turnbull,K.Drummond,Z.Schofield,Dr.E.Yates, Dr,S Guimond, Dr.Z.Huang, Dr.A.Powell, J.Henstock, Dr. T. Kinnunen
3 / ACTIVITY ASSESSED / Biotinylation of Heparin ( Fernig Method )
4 / MATERIALS INVOLVED / Attach copies of data sheet(s)
NAME / AMOUNT
max/stored / HAZARD / RISK PHRASES / HAZDAT NO*** / BIOSCIENCESNO***
Heparin / 42mg / N/A / N/A
EZ-Link NHS-LC-Biotin / 50mM / Irritant / R36 : irritating to the eyes
Water / 100ul / N/A / N/A
DMSO / 50ul / Irritant /
Combustible / R36/37/38: Irritating to eyes, respiratory system and skin
Ethanol / 100ml / Flammable / R11 : Highly Flammable
5 / INTENDED USE**
Give brief details and attach protocol/instructions
Heparin – compound to be biotinylated
Biotin – compound to be bound to heparin
DMSO –diluent for Biotin
Ethanol – Wash reagent
6 / RISKS to HEALTH and SAFETY from INTENDED USE
From personal exposure or hazardous reactions. Refer to OELs, flash points, etc., as appropriate. Are pregnant women, breast-feeding mothers especially at risk?
EZ-Link NHS-LC-Biotin – Risk of irritation to unprotected eyes.
DMSO – Risk of splashes to unprotected eyes and skin. Risk of fire at high temperatures.
Ethanol – Risk of fire.
7 / CONCLUSIONS ABOUT RISKS
Is level of risk acceptable? Can risk be prevented or reduced by change of substance/procedure? Are control measures necessary?
Due to the nature of some of the chemicals listed in this assessment there is a small risk . But due to the small amounts used and as long as measures laid out in section 8 are followed then the level of risk is acceptable. Non of the chemicals used can be replaced with other “ less hazardous” reagents.
8 / CONTROL MEASURES
Additional to Good Chemical Practice
Wearing of lab coat, chemical resistant gloves and eye protection.
Be aware of and avoid sources of ignition. Vapour may travel towards source of ignition.
To avoid inhalation of powdery substances wear a face mask when weighing out.
9 / INSTRUCTION/TRAINING
Specify course(s) and/or special arrangements.
One to one initial training
10 / MONITORING
Performance of control measures,
N/A
Personal exposure / Health Surveillance
N/A / N/A
11 / WASTE DISPOSAL PROCEDURE
See School Server for Approved Procedure Document on specific Chemical Waste Disposal.
All reagents can be disposed of down the sink with copious amounts of water.
12 / REVIEW
Enter the date or circumstances for review of assessment (maximum review interval 5 years)
20/08/07
13 / EMERGENCY ACTION
TO CONTROL HAZARDS / To stabilize situation eg spread absorbant on liquid spill; eliminate sources of ignition, etc.
Small spill of Ethanol or DMSO– extinguish all ignition sources, mop up with towel. Allow towel to dry in fume hood before disposal in the general contaminated waste bag.
Large spill of Ethanol or DMSO– Immediately extinguish ignition sources, evacuate immediate area. Wearing heavy rubber boots, respiratory equipment and rubber gloves spread sorbant material such as soda ash or dry-lime. Sweep up into a sealed container and place with stores for specialist waste disposal.
TO PROTECT PERSONNEL / Evacuation, protection for personnel involved in clean-up, Special First Aid
Extinguish all sources of ignition.
Evacuate immediate area for a large spill of DMSO or Ethanol. Wear self contained breathing apparatus, heavy rubber boots and gloves for the clean up.
Contact with reagents within this protocol require immediate washing in copious amounts of water. If discomfort persists then contact a physician.
TO RENDER SITE OF EMERGENCY SAFE / Clean-up/decontamination
Cleanse the area with a dilute solution of decon, dry the area thoroughly.
CONTACT PHONE
10.10.00
* Prefix T is used for Teaching Assessment Number.
** Please include amount of chemicals used and how.
***Hazdat No is the UNICOSHH datasheet report number.
Biosciences No is the Biosciences data sheet number.
UNICOSHH IS A CHEMICAL DATABASE ON THE HEALTH AND SAFETY UNIT SERVER.
BIOSCIENCES DATA SHEETS ARE AVAILABLE IN THE SCHOOL SAFETY OFFICE.
Biotinylation of Heparin ( Fernig Method )
Biotinylation of heparin on the free amino groups but also on N-sulphates.
Kinsella et al 1998 Glycoconjugate journal 15: 419-422
Method
1. Make 50mM solution of NHS-LC Biotin ( Pierce 21336 ) in DMSO.
( mw : 454.54, hence 22.7mg/ml = 50mM )
2. Make 42mg heparin in 100ul dd H2O
3. Add 50ul of 50mM biotin to the 100ul heparin solution ( or adjust the volumes accordingly )
4. Incubate overnight at room temperature.
5. Precipitate the heparin with 200ml of 100% Ethanol at -20C overnight. (add heparin to ethanol to precipitate better ) By morning ethanol should be misty with precipitated heparin.
6. Pour through a scint filter using a Buchner flask and vacuum pull.
7. Wash the filter with 50mls cold 100% Ethanol.
8. Solubalise the heparin from the scint filter with 4-5ml ddH2O.
9. Store at 4C until analysis.
10. Remove the free biotin by gel filtration using Hitrap desalting columns and FPLC reading absorbance
at 232nm.