The safe use and handling of cryogenic materials
Cryogenic materials are used in a vast majority of laboratories, especially wet labs. On a daily basis at the University of Florida, we receive shipments packed with dry ice, preserve samples with liquid nitrogen, remove impurities with cold traps or baths, and keep our equipment cooled with controlled internal environments. As with most things in the lab, all of these can be done safely if we recognize the hazards and work diligently to control them. If we become cavalier or lax, things can go very wrong. Unfortunately, there are documented cases where incidents with cryogens have resulted in serious injury and death. Some examples are:
A laboratory assistant died and four other people were injured in a chemical leak at a hospital in Edinburgh. The assistant died after liquid nitrogen spilled in a basement storage room.1
A university investigator was blinded in one eye when a cryotube exploded while being thawed. The probable cause was the rapid expansion of liquid nitrogen that had entered the tube through a small crack during storage. Suitable personal protective equipment for thawing cryotubes and handling cryogenic liquids consists of a face shield, heavy gloves, a buttoned lab coat, and pants or a long skirt. Cryotubes should be kept in a heavy, walled container or behind a safety shield while warming.2
A researcher at a university reported that a vial of potentially infectious materials “exploded” when she removed it from liquid nitrogen. As you may have guessed, the “explosion” occurred when the liquid N2 leaked into a vial and expanded when removed from the cold. This used to be a fairly common problem with heat-sealed glass ampules, because it was difficult to obtain perfectly fused glass with no microscopic holes.2
As we examine the literature, there are differing opinions about when a substance becomes a cryogenic material. For our discussion we will consider materials with boiling points below –75°C as cryogenic (so as to include dry ice).3
Cryogens are similar to other broad classes of chemicals because we can divide the concerns into physiological hazards and physical hazards. As with other hazardous chemicals in the lab, it should go without saying that the safety data sheet should be read and understood, and standard operating procedures should be developed and included in your chemical hygiene plan.
Within the physiological hazards category, we group the hazards into two main divisions: those that damage tissue from direct contact and those that can cause asphyxiation.
Those of us who are old enough and spent a lot of time in the sun in our youth are all too familiar with the effects of liquid nitrogen from our visits to the dermatologist. The quick, controlled spray of liquid nitrogen freezes and kills tissue in pretty short order. The same effect is true from accidental splashes or contact with these very cold materials. Therefore, the first rule is: Protect your skin and eyes.
Always wear safety glasses whenever you are near a cryogenic liquid or working with samples recently removed from cryogenic temperatures. In addition, also wear a full-face shield if a cryogenic liquid is being poured or if an open container of the cryogen may boil and splatter. To reduce the amount of splatter when transferring cryogenic liquids from one container to another, always start slowly and allow the vaporization to chill the receiving container before filling it. After the vaporization and liquid boiling have decreased, fill the container at the normal rate. 2
Cryogenic materials flow freely as do other liquids and as a result can splash and spill. It is important to wear liquid-resistant gloves to prevent splashed liquid from being absorbed and freezing the skin.
Cryogenic liquids contain a tremendous amount of potential gas volume. One unit volume of liquid nitrogen, for example, will expand to produce almost 700 unit volumes of gas when vaporized. This raises the concern of oxygen displacement. Normal air contains 19.5 percent oxygen by volume. One can begin to feel the effects of oxygen deficiency at about 18 percent, and sudden death may occur at about 6 percent. A leak or vessel breakage can result in an oxygen-deficient atmosphere rather quickly, especially in a small room with poor ventilation (e.g., an elevator or cold room). For example, an excerpt from an investigation reported by the American Industrial Hygiene Association stated, “Recently on the campus, a walk-in refrigerator was used to store dry ice. The dry ice was stored in a standard dry ice storage locker but the locker had been placed in the cold box to further reduce the rate of dry ice loss. The dry ice, of course, gave off carbon dioxide (CO2) gas as it sublimed, causing the refrigerator to build up CO2 levels of 12,000 parts per million (ppm)! In comparison, outdoor air contains only about 400 ppm CO2, and OSHA’s Permissible Exposure Limit for CO2 is 5,000 ppm.”3 We highly recommend developing specific procedures for storage and transport of all cryogens.
Within the physical hazards category, we can group the hazards into those that have an explosion risk from pressure buildup and those that have an explosion risk from chemical reactions.
Explosion — Pressure
As we mentioned above, the gas volume generated from the vaporization of the liquid phase is very large (e.g., liquid nitrogen expands almost 700 times when vaporized). If this phase change occurs in a vessel unable to contain the pressures exerted, it can fail dramatically. It is not uncommon to hear of lab-made cryotubes exploding when removed from storage. The liquid nitrogen can get into a cryotube through imperfect sealing and expands upon thawing while converting to the gas phase. Use tubes specifically designed for cryogenic storage and place them in a heavy-walled container or behind a safety shield while thawing. The tubes are also designed to be in the gas phase and not submerged in the liquid nitrogen in the storage Dewar. Overfilling a Dewar can cause sample tubes to be stored in the liquid phase, thus allowing liquid nitrogen to enter the tube.
Explosion — Chemical
Cryogenic fluids, such as nitrogen, with a boiling point below that of liquid oxygen are able to condense and accumulate oxygen from the atmosphere. Violent re-actions, for example rapid combustion or explosion, may occur if incompatible materials, such as most common organic compounds, come in contact with the oxygen. This might occur in an uncovered nitrogen trap used to condense out low boiling point liquids or an open Dewar flask. This is why it’s important to keep Dewar flasks covered with a loose-fitting cap. This prevents air and moisture from entering the container yet allows pressure to escape.
Here we have just scratched the surface on the hazards and controls associated with cryogens. Familiarity of these materials in the lab can lead to complacency, but these hazards can result in serious injury if not controlled. Anyone who handles or uses cryogenic liquids must have adequate knowledge of the particular material’s properties and the safe handling practices.4 Specific understanding should include:
- properties of the cryogen as a liquid, solid, or gas
- materials compatible for use with that cryogen (e.g., must be compatible with the temperatures and pressures of the material)
- protective equipment required and its proper use
- understanding of the equipment being used, including its safety devices
- emergency procedures, including first aid and treatment
1. Inquiry after man dies in chemical leak. BBC Online News, Monday, October 25, 1999. http://news.bbc.co.uk/1/hi/scotland/484813.stm
2. Lab Safety Cryogen Incidents. American Industrial Hygiene Association — Laboratory Health and Safety Committee. 2015. https://www.aiha.org/get-involved/VolunteerGroups/LabHSCommittee/Incident%20Pages/Lab-Safety-Cryogens-Incidents.aspx
3. Prudent Practice in the Laboratory: Handling and Disposal of Chemicals. National Academies Press, National Resource Council, Washington, D.C.1995. http://www.nap.edu/catalog/4911/prudent-practices-in-the-laboratory-handling-and-disposal-of-chemicals
4. How Do I Work Safely with Cryogenic Liquids? Canadian Centre for Occupational Health and Safety. Hamilton, ON, Canada. 2015. http://www.ccohs.ca/oshanswers/prevention/cryogens.html