DESCRIPTION
Phenol: Chloroform Extraction is a common laboratory technique used to separate proteins from nucleic acids. An equal volume of phenol: chloroform (50:50) is added to a nucleic acid sample. The mixture is centrifuged to separate the organic and aqueous phases. The organic phase is discarded. Ethyl ether is sometimes used to remove traces of chloroform.
HAZARDS ASSOCIATED WITH PHENOL:CHLOROFORM EXTRACTION
Biological Hazards: There are no biological hazards associated with this procedure unless human source materials are used.
Chemical Hazards: Phenol is readily absorbed through the skin and can cause severe burns to the eyes and skin. Phenol has a local anesthetic effect, so that no pain may be felt on initial contact. Systemic effects include damage to the liver and kidneys.
Chloroform is a skin and eye irritant, and it is a suspected human carcinogen and reproductive hazard. Adding chloroform to phenol enhances the ability of phenol to be absorbed by the skin.
Ethyl ether is a mild skin and eye irritant. The primary hazard associated with ethyl ether is that it is extremely flammable and it forms explosive peroxides after prolonged exposure to light and air. When kept for prolonged periods after it has been opened, ethyl ether forms peroxides that may explode when the container cap is removed or when it is concentrated during laboratory activities.
Refer to a material safety data sheet (MSDS) for more detailed information on these and other laboratory chemicals.
Equipment Hazards: Centrifugation may result in the formation of aerosols and droplets.
Special Work Practices
Perform all procedures involving greater than 500 ml of phenol or chloroform or any amount of ether in a chemical fume hood.
Wash hands thoroughly immediately after working with these chemicals. Never eat, drink, chew gum, apply cosmetics, take medicine or store food in a laboratory where these chemicals are used and stored.
Grasp both the tube and the cap when vortexing to prevent the cap from opening, causing a splash or aerosol.
Use sealed safety cups when centrifuging phenol: chloroform, and wait at least 10 minutes before opening the centrifuge to prevent exposure to aerosols.
Personal Protective Equipment
Eye Protection: Wear safety glasses when performing phenol: chloroform extraction, even when working in a fume hood.
Phenol can cause severe burns to the eyes. Wear chemical splash goggles when there is a splash hazard (for example, when pouring phenol).
Gloves: Wear disposable nitrile gloves to protect against accidental hand contact. If accidental contact occurs, remove and discard contaminated gloves immediately. (The breakthrough time for a 4 mil nitrile glove is approximately 3 minutes for chloroform.)
Protective Clothing: Wear standard laboratory apparel. (Lab coat, long pants and closed toe shoes.)
Emergency Procedures
Eye Contact: Immediately irrigate eyes at eyewash for 15 minutes, holding eyes open. Obtain medical attention.
Skin Contact: Immediately wash affected areas with soap and water. Remove contaminated clothing. Obtain medical attention.
Ingestion: Obtain medical attention immediately.
Inhalation: Seek medical attention if symptoms develop (wheezing, coughing, shortness of breath, burning in mouth, throat or chest).
Medical Attention: During working hours (8-5), staff should report to Occupation Medicine and students should report to Student Health Services. During evenings and weekends, staff and students should report to the emergency room.
Storage
Store ethyl ether in a flammable liquid storage cabinet. Store phenol away from strong oxidizers and store chloroform away from strong alkalis. Store chloroform in a dark location.
Write the date received and the date opened on all containers of ether. Discard open containers of ether within six months of opening.
Waste Disposal
Discard phenol chloroform mixture in a properly labeled waste carboy. Attach a white hang tag marked "Phenol Chloroform" to the handle of the carboy. All waste containers must be in secondary containment. Hang tags, labels, waste carboys and secondary containment containers are available through EHRS. Discard all syringes used in phenol: chloroform extraction in a sharps container labeled "Mutagen/Carcinogen Sharps Do Not Autoclave."
Place all pipettes that contacted phenol chloroform in a rigid container and discard as hazardous waste.
Contact EHRS to arrange for disposal.
Spill Procedures
Laboratory personnel should clean up small spills in the fume hood. Wear Viton or Silver Shield/4H gloves when cleaning up a spill. (Do not wear nitrile gloves due to the risk of direct or prolonged contact when cleaning up a spill.) Place absorbent materials used to clean up the spill in plastic bags and discard as hazardous waste.
For spills greater than 500 ml outside a fume hood, evacuate the laboratory and contact EHRS.