Liquid nitrogen and cryogenic storage: temperature, vapour phase and safety

Liquid nitrogen boils at about −196 °C (about −320 °F, 77 K) at normal pressure (NIST Chemistry WebBook), which makes it the coldest material most labs handle. It is used to keep living cells and tissue for years, to freeze samples in seconds, and to cool instruments. This guide explains what it is, how cold it is, how cryogenic storage works and what makes it hazardous. It also explains why freeze-dried peptides don't need it.

Terms are defined in the glossary.

What is liquid nitrogen?

Nitrogen makes up about 78% of the air. Cooled far enough, it condenses into a clear, colourless liquid that looks like water. It is made by cooling and compressing air, then separating the nitrogen from the oxygen and argon by distillation.

Liquid nitrogen is inert: it doesn't burn and doesn't react with samples. That, its low cost and its very low temperature make it the standard coolant for cryogenic work.

Sources: NIST Chemistry WebBook and BCGA CP30.

Liquid nitrogen temperature

Liquid nitrogen at normal pressure sits at its boiling point, about −196 °C. It can't get warmer while it is still liquid: heat from the room makes it boil off rather than warm up. That is why an open Dewar of liquid nitrogen gently steams all the time.

Can you touch liquid nitrogen?

Not safely. When something warm is dropped into it, the liquid boils so fast that a layer of gas forms around the object and insulates it. This is called the Leidenfrost effect. It is why a sample plunged into liquid nitrogen fizzes for a moment, and why small splashes skitter across a bench. That brief protection fails as soon as liquid is held against skin, which causes cold burns; see cold burns below.

Liquid nitrogen vs dry ice

Both turn into a large volume of gas, so neither should be sealed in an airtight container. For dry ice in shipping, see why freeze-dried peptides ship at room temperature.

Why labs store samples so cold

Every chemical and biological process slows down as temperature drops. For most purposes, a −20 °C or −80 °C freezer is cold enough. Some samples need more:

• Living cells and tissue. At the very low temperatures of liquid nitrogen storage, the water in and around cells is locked in a glassy state where almost no change can happen. Cells kept below that point can be stored for many years and revived. A −80 °C freezer isn't cold enough for this over the long term.
• Samples for long-term archives, where any slow change over decades matters.
• Snap-freezing. Plunging a sample into liquid nitrogen freezes it within seconds, which forms smaller ice crystals and limits damage.

See freezer temperatures for sample storage for the common storage temperatures and what each is used for. For how temperature controls the rate of breakdown, see the Arrhenius equation.

How cryogenic storage works

Samples are kept in a Dewar or a larger cryogenic storage vessel: a container with a vacuum between two walls, like a large vacuum flask. The vacuum slows the heat getting in, so the liquid nitrogen boils off slowly and is topped up on a schedule.

There are two ways to store samples in it.

Vapour-phase storage is now more common. Samples sit in racks above a pool of liquid at the bottom, or in a vessel whose walls are cooled by liquid. They stay far colder than any mechanical freezer without being immersed.

Cryovials

Samples go into cryovials: small, thick-walled polypropylene tubes with screw caps, made to survive the cold. Glass vials and ordinary plastic tubes can crack. Cryovials stored in the liquid phase can take in liquid nitrogen through the cap seal. When they are taken out and warm up, the trapped liquid turns to gas about 683 times its volume, and the vial can burst. Vapour-phase storage, or sealing vials in an outer sleeve, avoids this.

How long does liquid nitrogen last in a Dewar?

It depends on the size and design of the vessel and how often it is opened. Makers quote a static hold time: how long a full, closed Dewar lasts before it is empty. For example, one 35-litre storage Dewar is rated at about 152 days, or roughly 0.23 litres boiled off a day; the maker notes these are nominal figures that vary with the contents, the surroundings and how the vessel is used (IC Biomedical LD35, via BSI). Opening the lid and removing racks shortens it, so vessels are topped up on a schedule.

The hazards of liquid nitrogen

Liquid nitrogen is not toxic, but it has three serious hazards. The British Compressed Gases Association sets out safe practice in its code of practice for Dewars (BCGA CP30).

Asphyxiation

As liquid nitrogen boils off, it adds nitrogen to the air and lowers the share of oxygen. There is no smell or warning. In a small or poorly ventilated room, such as a cold room, a lift or a storage cupboard, oxygen can fall to a dangerous level. Cold nitrogen gas is dense and collects in low areas.

Labs control this by:

• keeping storage and filling areas well ventilated,
• fitting oxygen depletion monitors with alarms where large amounts are kept,
• never travelling in a lift with a Dewar, and sending it up or down on its own, and
• assessing the quantity against the room size before any work starts.

Cold burns

Contact with liquid nitrogen, or with metal it has cooled, freezes skin in moments. Short splashes often roll off because of the Leidenfrost effect, but liquid trapped against skin by a glove, sleeve or watch strap causes deep burns. Standard protection is a full face shield, loose-fitting cryogenic gloves that can be shaken off, a lab coat and closed shoes.

Pressure

Because the liquid expands so much as it turns to gas, it must never be sealed in a closed container. A tightly capped bottle or flask can burst. Dewars have loose-fitting lids or pressure-relief valves for this reason.

Oxygen enrichment

Oxygen boils at a slightly higher temperature than nitrogen, about −183 °C (90.2 K, NIST Chemistry WebBook). Surfaces cooled by liquid nitrogen can condense oxygen from the air, producing a pale blue liquid rich in oxygen. That makes nearby materials burn far more easily. It is one reason open Dewars aren't left uncovered for long.

Do freeze-dried peptides need liquid nitrogen?

No. Freeze-dried (lyophilised) peptides are dry solids. With almost no water present, the reactions that break them down are already very slow, and a −20 °C freezer is the standard long-term condition. Bachem, a large peptide manufacturer, advises keeping freeze-dried peptides below −15 °C in a tightly closed container (Bachem). Liquid nitrogen adds cost and hazard without a meaningful gain for a sealed, dry vial. See how to store freeze-dried peptides.

We store freeze-dried peptides at −20 °C, sealed and protected from light. Each product page lists the recommended storage condition for that compound.

Related guides

For other storage temperatures and how to monitor a freezer, see freezer temperatures for sample storage. For why freeze-dried peptides can travel without cold packs or dry ice, see why lyophilised peptides ship at room temperature. For the law on hazardous substances, see COSHH and safety data sheets. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.