Lyophilization/freeze drying is a method of extracting the water from Biological samples,foods and other products so that foods or products remain stable and are easier to store at room temperature. Biological materials should be dried to stabilize them for storage, preservation and shipping. In many cases this drying can cause damage and some loss of cellular or protein activity. Lyophilization significantly reduces damage to biological samples.

Principle of Lyophilization

Lyophilization is based on a simple principle of physics called “SUBLIMATION”. Sublimation is the process of transition of a substance from solid to the vapor state without passing through an intermediate liquid phase. The process of lyophilization consists of:

• Freezing of the product to convert the water in the product to ice form,
• Sublimation of ice directly into water vapor under vacuum.
• Drawing off the water vapor
• Once the ice has been sublimated, the products are freeze-dried and can be removed from machine.

The principle advantages of lyophilization as a drying process are:

• Minimum damage and loss of activity in delicate heat-liable materials
• Speed and completeness of rehydration
• Possibility of accurate, clean dosing into final product containers
• Porous, friable structure

The principle disadvantages of lyophilization are:

• High capital cost of equipment (about three times more than other methods)
• High energy costs (2-3 times more than other methods)
• Long process time (typically 24 hour drying cycle)

Equipment used for Lyophilization – LYOPHILIZER

A lyophilizer consists of

• A vacuum chamber containing product shelves which are capable of cooling and heating containers and their contents.
• A vacuum pump, a refrigeration unit, which is associated controls are connected to the vacuum chamber.

Samples are generally placed in containers such as glass vials that are placed on the shelves within the vacuum chamber.

Cooling elements within the shelves freeze the product. Once the product is frozen, the vacuum pump evacuates the chamber and the product is heated. Heat is transferred by thermal conduction from the shelf, through the vial, and ultimately into the product.

Comparison with Liquid-Phase drying

Lyophilization avoid denaturation that is caused by heating the product, by maintaining it frozen throughout drying. This is the most obvious advantage over liquid-phase drying which cause the denaturation of proteins and other products.

Equally important is that in liquid-phase drying there is an undesirable shrinkage and concentration of active constituents that causes damage as well as a movement of these constituents to the surface of evaporation, where they form a dense, impermeable skin that inhibits drying, and later, rehydration. Such effects can be avoided by spray drying, but this requires brief exposure to temperatures around 100 degree C.

Advantages of the Lyophilization

Lyophilization has many advantages over the other drying and preserving techniques.

1. It maintains food/ biochemical and chemical reagent quality because they remains at a temperature that is below the freezing-point during the process of sublimation.The use of lyophilization is particularly important when processing lactic bacteria, because these products are easily affected by heat.
2. Food/biochemicals and chemical reagents which are lyophilized can usually be stored without refrigeration, which results in a significant reduction of storage and transportation costs.
3. Lyophilization greatly reduces weight, and this makes the products easier to transport. For example, many foods contain as much as 90% water. These foods are 10 times lighter after lyophilization.
4. Because they are porous, most freeze-dried products can be easily rehydrated. Lyophilization does not significantly reduce volume, therefore water quickly regains its place in the molecular structure of the food/ biochemicals and chemical reagents.

Safety Guidelines for Lyophilizer

• Mammalian cells can not be preserved by lyophilization because it can desteoy mammalian cells. Many microorganisms and proteins survive lyophilization well,because they rehydrate easily and quickly because of the porous structure left after the ice sublimes.
• Wear appropriate eye protection at all times when working with or near a lyophilizer.
• Specimens shell-frozen in ampoules are dried on a vacuum manifold or in a chamber-type drier at low negative pressure. If the glass neck of the ampoule is sealed off while the ampoule is still under vacuum, it may cause implosion, either during the sealing or later when the evacuated ampoule is being opened. To avoid this, after drying is completed, and before sealing is done, bring the pressure within the ampoule back to normal by gradually introducing dry nitrogen, avoiding turbulent disturbance of the dry product.
• The narrow or constricted neck of the ampoule is contaminated if the specimen is allowed to run down the wall of the neck during filling. Subsequently, when the ampoule is sealed with a torch, the dried material on the wall becomes charred or partially decomposed; residues of this material may adversely affect the dried material when it is reconstituted. To avoid this, a syringe with a long cannula or a Pasteur-type pipette should be used to fill the vial. Do not allow the delivery end of the cannula or pipette to touch the neck of the vial.
• All ampoules used for freeze-drying of cultures, toxins, or other biohazardous material should be fabricated of Pyrex-type glass. This type of glass requires a high-temperature torch using an air-gas or oxygen-gas mixture for sealing. These hard glass ampoules are much less apt to form gas bubbles that burst inwardly during sealing under vacuum than the soft glass ampoules and are more resistant to breakage during handling and storage.
• The filling of ampoules and vials with infectious specimens, the subsequent freeze-drying, and sealing or closing of ampoules and vials in the preparation of dry infectious specimens should be performed in a biological safety cabinet. The same is true for the preparation of ampoules and vials containing liquid specimens not subject to freeze-drying.

Safety precautions to be taken will depend on the agents, equipment, and containment available.

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