5.5. Lyophilisation (freeze-drying)

Protein (or any other non-volatile molecule) samples can be concentrated by evaporating water and other volatile compounds from the sample. In principle, this could be achieved by simply heating the sample. However, most proteins would unfold during such a simple evaporation process. In order to prevent the denaturation of proteins, the sample is transferred into a glass container and is frozen as quickly as possible, usually by immersing the outside of the container into liquid nitrogen. Moreover, the container is rotated in order to spread and freeze the sample on a large surface area. The glass container with the sample is then placed into an extremely low-pressure space (vacuum) that contains a cooling coil as well. The cooling coil acts as a condenser. The temperature of the coil is usually lower than -50°C. Volatile compounds of the frozen sample will evaporate (sublimate) in the vacuum. The process of evaporation (in this case, sublimation) absorbs heat. This effect keeps the sample frozen. Evaporated molecules are captured from the gas phase by the cooling coil, forming a frozen layer on it. At the end of the process, proteins and other non-volatile compounds of the sample remain in the container in a solid form. This process does not cause irreversible denaturation of proteins. Thus, it is a method frequently used not only to concentrate but also to preserve proteins or other sensitive biomolecules for long-term storage. Such samples can usually be stored for years without a significant deterioration of quality. However, before lyophilisation it is very important to carefully consider all non-volatile compounds of the initial sample as these will concentrate along with the proteins. Non-volatile acids or bases can cause extreme pH, and the presence of salts can result in very high ionic strength when the sample is resolubilised.